Search

Ribonucleotide reductases (RNRs) are the sole de novo source of deoxyribonucleotides for DNA synthesis and repair across all organisms and carry out their reaction via a radical mechanism. RNR from Chlamydia trachomatis generates its turnover-initiating cysteinyl radical by long-range reduction of a Mn(IV)/Fe(III) cofactor, producing a Mn(III)/Fe(III) intermediate. Herein, we characterize the protonation states of the inorganic ligands in this reduced state using advanced pulse electron paramagnetic resonance (EPR) spectroscopy and 2 H-isotope labeling. A strongly coupled deuteron is observed by hyperfine sublevel correlation (HYSCORE) spectroscopy experiments and indicates the presence of a bridging hydroxo ligand. Isotope-dependent EPR line broadening analysis and the magnitude of the estimated Mn-Fe exchange coupling constant together suggest a μ-oxo/μ-hydroxo core. Two distinct signals detected in electron-nuclear double resonance (ENDOR) spectra are attributable to less strongly coupled hydrons of a terminal water ligand to Mn(III). Together, these experiments imply that the reduced cofactor has a mixed μ-oxo/μ-hydroxo core with a terminal water ligand on Mn(III). This structural assignment sheds light generally on the reactivity of Mn/Fe heterobimetallic sites and, more specifically, on the proton-coupling in the electron transfer that initiates ribonucleotide reduction in this subclass of RNRs.

I present the perspective that the divalent metalome and the metabolome can be modeled as a network of chelating interactions instead of separate entities. I review progress in understanding the complex cellular environment, in particular recent contributions to modeling metabolite-Mg 2+ interactions. I then demonstrate a simple extension of these strategies based approximately on intracellular Escherichia coli concentrations. This model is composed of four divalent metal cations with a range of cellular concentrations and physical properties (Mg 2+ , Ca 2+ , Mn 2+ , and Zn 2+ ), eight representative metabolites, and interaction constants. I applied this model to predict the speciation of divalent metal cations between free and metabolite-chelated species. This approach reveals potentially beneficial properties, including maintenance of free divalent metal cations at biologically relevant concentrations, buffering of free divalent metal cations, and enrichment of functional metabolite-chelated species. While currently limited by available interaction coefficients, this modeling strategy can be generalized to more complex systems. In summary, biochemists should consider the potential of cellular metabolites to form chelating interactions with divalent metal cations.

N -Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1- exo -acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H · abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H · from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2 H 2 O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S] 1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans , for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Parkinson's disease (PD) is characterized by the toxic oligomeric and fibrillar phases formed by monomeric alpha-synuclein (α-syn). Certain nanoparticles have been demonstrated to promote protein aggregation, while other nanomaterials have been found to prevent the process. In the current work, we use nuclear magnetic resonance spectroscopy in conjunction with isothermal titration calorimetry to investigate the cause and mechanism of these opposing effects at the amino acid protein level. The interaction of α-syn with two types of nanomaterials was considered: citrate-capped gold nanoparticles (AuNPs) and graphene oxide (GO). In the presence of AuNPs, α-syn aggregation is accelerated, whereas in the presence of GO, aggregation is prevented. The study indicates that GO sequesters the NAC region of α-syn monomers through electrostatic and hydrophobic interactions, leading to a reduced elongation rate, and AuNPs leave the NAC region exposed while binding the N-terminus, leading to higher aggregation. The protein's inclination toward quicker aggregation is explained by the binding of the N-terminus of α-syn with the gold nanoparticles. Conversely, a comparatively stronger interaction with GO causes the nucleation and growth phases to be postponed and inhibits intermolecular interactions. Our finding offers novel experimental insights at the residue level regarding the aggregation of α-syn in the presence of various nanomaterials and creates new opportunities for the development of suitably functionalized nanomaterial-based therapeutic reagents against Parkinson's and other neurodegenerative diseases.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA] 25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA] 25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA] 25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

Small nucleolytic ribozymes are RNAs that cleave their own phosphodiester backbone. While proteinaceous enzymes are regulated by a variety of known mechanisms, methods of regulation for ribozymes remain unclear. Twister is one ribozyme class for which many structural and catalytic properties have been elucidated. However, few studies have analyzed the activity of twister ribozymes in the context of a native flanking sequence, even though ribozymes as transcribed in nature do not exist in isolation. Interactions between the ribozyme and its neighboring sequences can induce conformational changes that inhibit self-cleavage, providing a regulatory mechanism that could naturally determine ribozyme activity in vivo and in synthetic applications. To date, eight twister ribozymes have been identified within the staple crop rice ( Oryza sativa ). Herein, we select several twister ribozymes from rice and show that they are differentially regulated by their flanking sequence using published RNA-seq data sets, structure probing, and cotranscriptional cleavage assays. We found that the Osa 1-2 ribozyme does not interact with its flanking sequences. However, sequences flanking the Osa 1-3 and Osa 1-8 ribozymes form inactive conformations, referred to here as "ribozymogens", that attenuate ribozyme self-cleavage activity. For the Osa 1-3 ribozyme, we show that activity can be rescued upon addition of a complementary antisense oligonucleotide, suggesting ribozymogens can be controlled via external signals. In all, our data provide a plausible mechanism wherein flanking sequence differentially regulates ribozyme activity in vivo . More broadly, the ability to regulate ribozyme behavior locally has potential applications in control of gene expression and synthetic biology.

MppQ is an enzyme of unknown function from Streptomyces hygroscopicus (ShMppQ) that operates in the biosynthesis of the nonproteinogenic amino acid L-enduracididine (L-End). Since L-End is a component of several peptides showing activity against antibiotic-resistant pathogens, understanding its biosynthetic pathway could facilitate the development of chemoenzymatic routes to novel antibiotics. Herein, we report on the crystal structures of ShMppQ complexed with pyridoxal-5'-phosphate (PLP) and pyridoxamine-5'-phosphate (PMP). ShMppQ is similar to fold-type I PLP-dependent aminotransferases like aspartate aminotransferase. The tertiary structure of ShMppQ is composed of an N-terminal extension, a large domain, and a small domain. The active site is placed at the junction of the large and small domains and includes residues from both protomers of the homodimer. We also report the first functional characterization of MppQ, which we incubated with the enzymatically produced 2-ketoenduracidine and observed the conversion to L-End, establishing ShMppQ as the final enzyme in L-End biosynthesis. Additionally, we have observed that MppQ has a relatively high affinity for 2-keto-5-guanidinovaleric acid (i.e., 2-ketoarginine), a shunt product of MppP, indicating the potential role of MppQ in increasing the efficiency of L-End biosynthesis by converting 2-ketoarginine back to the starting material, l-arginine. A panel of potential amino-donor substrates was tested for the transamination activity against a saturating concentration of 2-ketoarginine in end-point assays. Most l-Arg was produced with l-ornithine as the donor substrate. Steady-state kinetic analysis of the transamination reaction with l-Orn and 2-ketoarginine shows that the kinetic constants are in line with those for the amino donor substrate of other fold-type I aminotransferases.

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl - or Br - ), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O 2 . Halide, 2OG, and (lastly) O 2 all coordinate directly to the cofactor to initiate its conversion to a cis -halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H • ) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O 2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

Bacteria that infect the human gut must compete for essential nutrients, including iron, under a variety of different metabolic conditions. Several enteric pathogens, including Vibrio cholerae and Escherichia coli O157:H7, have evolved mechanisms to obtain iron from heme in an anaerobic environment. Our laboratory has demonstrated that a radical S- adenosylmethionine (SAM) methyltransferase is responsible for the opening of the heme porphyrin ring and release of iron under anaerobic conditions. Furthermore, the enzyme in V. cholerae , HutW, has recently been shown to accept electrons from NADPH directly when SAM is utilized to initiate the reaction. However, how NADPH, a hydride donor, catalyzes the single electron reduction of a [4Fe-4S] cluster, and/or subsequent electron/proton transfer reactions, was not addressed. In this work, we provide evidence that the substrate, in this case, heme, facilitates electron transfer from NADPH to the [4Fe-4S] cluster. This study uncovers a new electron transfer pathway adopted by radical SAM enzymes and further expands our understanding of these enzymes in bacterial pathogens.

The diheme bacterial cytochrome c peroxidase (bC c P)/MauG superfamily is a diverse set of enzymes that remains largely uncharacterized. One recently discovered member, MbnH, converts a tryptophan residue in its substrate protein, MbnP, to kynurenine. Here we show that upon reaction with H 2 O 2 , MbnH forms a bis -Fe(IV) intermediate, a state previously detected in just two other enzymes, MauG and BthA. Using absorption, Mössbauer, and electron paramagnetic resonance (EPR) spectroscopies coupled with kinetic analysis, we characterized the bis -Fe(IV) state of MbnH and determined that this intermediate decays back to the diferric state in the absence of MbnP substrate. In the absence of MbnP substrate, MbnH can also detoxify H 2 O 2 to prevent oxidative self damage, unlike MauG, which has long been viewed as the prototype for bis -Fe(IV) forming enzymes. MbnH performs a different reaction from MauG, while the role of BthA remains unclear. All three enzymes can form a bis -Fe(IV) intermediate but within distinct kinetic regimes. The study of MbnH significantly expands our knowledge of enzymes that form this species. Computational and structural analyses indicate that electron transfer between the two heme groups in MbnH and between MbnH and the target tryptophan in MbnP likely occurs via a hole-hopping mechanism involving intervening tryptophan residues. These findings set the stage for discovery of additional functional and mechanistic diversity within the bC c P/MauG superfamily.

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg 2+ under conditions that mimic the Escherichia coli cytoplasm. We determined Mg 2+ binding constants for the top 15 E. coli metabolites, comprising 80% of the metabolome by concentration at physiological pH and monovalent ion concentrations. These data were used to inform the development of an artificial cytoplasm that mimics in vivo E. coli conditions, which we term "Eco80". We empirically determined that the mixture of E. coli metabolites in Eco80 approximated single-site binding behavior toward Mg 2+ in the biologically relevant free Mg 2+ range of ∼0.5 to 3 mM Mg 2+ , using a Mg 2+ -sensitive fluorescent dye. Effects of Eco80 conditions on the thermodynamic stability, chemical stability, structure, and catalysis of RNA were examined. We found that Eco80 conditions lead to opposing effects on the thermodynamic and chemical stabilities of RNA. In particular, the thermodynamic stability of RNA helices was weakened by 0.69 ± 0.12 kcal/mol, while the chemical stability was enhanced ∼2-fold, which can be understood using the speciation of Mg 2+ between weak and strong Mg 2+ -metabolite complexes in Eco80. Overall, the use of Eco80 reflects RNA function in vivo and enhances the biological relevance of mechanistic studies of RNA.

Eukaryotic gene compaction takes place at multiple levels to package DNA to chromatin and chromosomes. Two of the most fundamental levels of DNA packaging are at the nucleosome and dinucleosome stacks. The nucleosome is the basic gene-packing unit and is composed of DNA wrapped around a histone core. Nucleosomes stack with one another for further compaction of DNA. The first stacking step leads to dinucleosome formation, which is driven by internucleosomal interactions between various parts of two nucleosomes. Histone proteins are rich targets for post-translational modifications, some of which affect the structure of the nucleosome and the interactions between nucleosomes. These effects are often implicated in the regulation of various genomic transactions. In particular, histone H2B ubiquitylation has been associated with facilitated transcription and hexasome formation. Here, we employed semi-synthetically ubiquitylated histone H2B and single-molecule FRET to investigate the effects of H2B ubiquitylations at lysine 34 (H2BK34) and lysine 120 (H2BK120) on the structure of the nucleosome and the interactions between two nucleosomes. Our results suggest that H2BK34 ubiquitylation widens the DNA gyre gap in the nucleosome and stabilizes long- and short-range internucleosomal interactions while H2BK120 ubiquitylation does not affect the nucleosome structure or internucleosomal interactions. These results suggest potential roles for H2B ubiquitylations in facilitated transcription and hexasome formation while maintaining the structural integrity of chromatin.

The enzyme BesC from the β - e thynyl-l- s erine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-l-lysine (produced from l-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-l-allylglycine and can analogously fragment l-Lys itself. BesC belongs to the emerging family of O 2 -activating non-heme-diiron enzymes with the "heme-oxygenase-like" protein fold (HDOs). Here, we show that the binding of l-Lys or an analogue triggers capture of O 2 by the protein's diiron(II) cofactor to form a blue μ-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino- N -oxygenase domain of SznF. The ∼5- and ∼30-fold faster decay of the intermediate in reactions with 4-thia-l-Lys and (4 RS )-chloro-dl-lysine than in the reaction with l-Lys itself and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of l-allylglycine in the reaction with 4,4,5,5-[ 2 H 4 ]-l-Lys suggest that the peroxide intermediate or a reversibly connected successor complex abstracts a hydrogen atom from C4 to enable olefin formation. Surprisingly, the sluggish substrate l-Lys can dissociate after triggering intermediate formation, thereby allowing one of the better substrates to bind and react. The structure of apo BesC and the demonstrated linkage between Fe(II) and substrate binding suggest that the triggering event involves an induced ordering of ligand-providing helix 3 (α3) of the conditionally stable HDO core. As previously suggested for SznF, the dynamic α3 also likely initiates the spontaneous degradation of the diiron(III) product cluster after decay of the peroxide intermediate, a trait emerging as characteristic of the nascent HDO family.

Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O 2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multiscale simulations, that positively charged residues generate large forces that move the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but move the A- and P-sites closer together. These conformational changes, respectively, increase and decrease the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation and provide a framework for interpreting experimental results on translation speed.

G-Quadruplexes (GQs) are compact, stable structures in DNA and RNA comprised of two or more tiers of quartets whose G-rich motif of tracts of two or more G's occurs commonly within genomes and transcriptomes. While thermodynamically stable in vitro , these structures remain difficult to study in vivo . One approach to understanding GQ in vivo behavior is to test whether conditions and molecules found in cells facilitate their folding. Polyamines are biogenic polycations that interact with RNA. Among common polyamines, spermine contains the highest charge and is found in eukaryotes, making it a good candidate for association with high-charge density nucleic acid structures like GQs. Using a variety of techniques, including ultraviolet-detected thermal denaturation, circular dichroism, size exclusion chromatography, and confocal microscopy, on an array of quadruplex sequence variants, we find that eukaryotic biological concentrations of spermine induce microaggregation of three-tiered G-rich sequences, but not of purely two-tiered structures, although higher spermine concentrations induce aggregation of even these. The formation of microaggregates can also be induced by addition of as little as a single G to a two-tiered structure; moreover, they form at biological temperatures, are sensitive to salt, and can form in the presence of at least some flanking sequence. Notably, GQ aggregation is not observed under prokaryotic-like conditions of no spermine and higher NaCl concentrations. The sequence, polyamine, and salt specificity of microaggregation reported herein have implications for the formation and stability of G-rich nucleic acid aggregates in vivo and for functional roles for understudied GQ sequences with only two quadruplex tiers.

RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary structures in which its negatively charged ribose-phosphate backbone interacts with metal ions. Magnesium, the most abundant divalent metal ion in cells, neutralizes the backbone, thereby playing essential roles in RNA folding and function. This has been known for more than 50 years, and there are now thousands of in vitro studies, most of which have used ≥10 mM free Mg 2+ ions to achieve optimal RNA folding and function. In the cell, however, concentrations of free Mg 2+ ions are much lower, with most Mg 2+ ions chelated by metabolites. In this Perspective, we curate data from a number of sources to provide extensive summaries of cellular concentrations of metabolites that bind Mg 2+ and to estimate cellular concentrations of metabolite-chelated Mg 2+ species, in the representative prokaryotic and eukaryotic systems Escherichia coli , Saccharomyces cerevisiae , and iBMK cells. Recent research from our lab and others has uncovered the fact that such weakly chelated Mg 2+ ions can enhance RNA function, including its thermodynamic stability, chemical stability, and catalysis. We also discuss how metabolite-chelated Mg 2+ complexes may have played roles in the origins of life. It is clear from this analysis that bound Mg 2+ should not be simply considered non-RNA-interacting and that future RNA research, as well as protein research, could benefit from considering chelated magnesium.

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered reverse transcriptase that "jumps" across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.

A recently described DNA polymerase ribozyme, obtained by in vitro evolution, provides the opportunity to investigate mechanistic features of RNA catalysis using methods that previously had only been applied to DNA polymerase proteins. Insight can be gained into the transition state of the DNA polymerization reaction by studying the behavior of various β,γ-bridging substituted methylene (CXY; X, Y = H, halo, methyl) or imido (NH) dNTP analogues that differ with regard to the p K a4 of the bisphosphonate or imidodiphosphate leaving group. The apparent rate constant ( k pol ) of the polymerase ribozyme was determined for analogues of dGTP and dCTP that span a broad range of acidities for the leaving group, ranging from 7.8 for the CF 2 -bisphosphonate to 11.6 for the CHCH 3 -bisphosphonate. A Brønsted plot of log( k pol ) versus p K a4 of the leaving group demonstrates linear free energy relationships (LFERs) for dihalo-, monohalo-, and non-halogen-substituted analogues of the dNTPs, with negative slopes, as has been observed for DNA polymerase proteins. The unsubstituted dNTPs have a faster catalytic rate than would be predicted from consideration of the linear free energy relationship alone, presumably due to a relatively more favorable interaction of the β,γ-bridging oxygen within the active site. Although the DNA polymerase ribozyme is considerably slower than DNA polymerase proteins, it exhibits a similar LFER fingerprint, suggesting mechanistic commonality pertaining to the buildup of negative charge in the transition state, despite the very different chemical compositions of the two catalysts.

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA-damaging agents and is tightly regulated to restrict TLS to damaged DNA. Replication protein A (RPA), the major single-strand DNA (ssDNA) binding protein complex, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single-molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18·RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination and represent, to the best of our knowledge, the first example of ATP-independent translocation of a protein complex along a protein filament.

l-Asparaginase (EC 3.5.1.1) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of l-asparaginase. First, a type II bacterial variant of l-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, l-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the l-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of l-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful l-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer l-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.

Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector. We utilized variable temperature isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments to better understand the entropic contributions to allosteric effector binding, including changes to solvent entropy and protein conformational entropy. Upon binding either Tyr or Trp, ScCM experiences a quenching of motions on the picosecond-to-nanosecond time scale, which we could relate to a loss of protein conformational entropy. Further ITC and NMR studies were consistent with the Tyr-bound form of ScCM being associated with more water molecules compared to the Trp-bound form and Tyr binding being associated with a less positive solvent entropy change. These studies provide insight into the role of structural dynamics in ScCM function and highlight the importance of solvent entropy changes in allosteric regulation, a historically underappreciated concept.

Antibiotic resistance continues to spread at an alarming rate, outpacing the introduction of new therapeutics and threatening to globally undermine health care. There is a crucial need for new strategies that selectively target specific pathogens while leaving the majority of the microbiome untouched, thus averting the debilitating and sometimes fatal occurrences of opportunistic infections. To address these challenges, we have adopted a unique strategy that focuses on oxygen-sensitive proteins, an untapped set of therapeutic targets. MqnE is a member of the radical S -adenosyl-l-methionine (RS) superfamily, all of which rely on an oxygen-sensitive [4Fe-4S] cluster for catalytic activity. MqnE catalyzes the conversion of didehydrochorismate to aminofutalosine in the essential menaquinone biosynthetic pathway present in a limited set of species, including the gut pathogen Helicobacter pylori ( Hp ), making it an attractive target for narrow-spectrum antibiotic development. Indeed, we show that MqnE is inhibited by the mechanism-derived 2-fluoro analogue of didehydrochorismate (2F-DHC) due to accumulation of a radical intermediate under turnover conditions. Structures of MqnE in the apo and product-bound states afford insight into its catalytic mechanism, and electron paramagnetic resonance approaches provide direct spectroscopic evidence consistent with the predicted structure of the radical intermediate. In addition, we demonstrate the essentiality of the menaquinone biosynthetic pathway and unambiguously validate 2F-DHC as a selective inhibitor of Hp growth that exclusively targets MqnE. These data provide the foundation for designing effective Hp therapies and demonstrate proof of principle that radical SAM proteins can be effectively leveraged as therapeutic targets.

Specifier proteins (SPs) are components of the glucosinolate-myrosinase defense system found in plants of the order Brassicales (brassicas). Glucosinolates (GLSs) comprise at least 150 known S -(β-d-glucopyranosyl)thiohydroximate- O -sulfonate compounds, each with a distinguishing side chain linked to the central carbon. Following tissue injury, the enzyme myrosinase (MYR) promiscuously hydrolyzes the common thioglycosidic linkage of GLSs to produce unstable aglycone intermediates, which can readily undergo a Lossen-like rearrangement to the corresponding organoisothiocyanates. The known SPs share a common protein architecture but redirect the breakdown of aglycones to different stable products: e pithionitrile ( E SP), n itrile ( N SP), or t hiocyanate ( T FP). The different effects of these products on brassica consumers motivate efforts to understand the defense response in chemical detail. Experimental analysis of SP mechanisms is challenged by the instability of the aglycones and would be facilitated by knowledge of their lifetimes. We developed a spectrophotometric method that we used to monitor the rearrangement reactions of the MYR-generated aglycones from nine GLSs, discovering that their half-lives ( t 1/2 ) vary by a factor of more than 50, from <3 to 150 s (22 °C). The t 1/2 of the sinigrin-derived allyl aglycone (34 s), which can form the epithionitrile product (1-cyano-2,3-epithiopropane) in the presence of ESP, proved to be sufficient to enable spatial and temporal separation of the MYR and ESP reactions. The results confirm recent proposals that ESP is an autonomous iron-dependent enzyme that intercepts the unstable aglycone rather than a direct effector of MYR. Knowledge of aglycone lifetimes will enable elucidation of how the various SPs reroute aglycones to different products.

Iron is essential for nearly every organism, and mismanagement of its intracellular concentrations (either deficiency or excess) contributes to diminished virulence in human pathogens, necessitating intricate metalloregulatory mechanisms. To date, although several metal-responsive riboswitches have been identified in bacteria, none has been shown to respond to Fe II . The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to Ni II and Co II but thought not to respond to Fe II , on the basis of aerobic, in vitro assays; its function in vivo is not well understood. We constructed a fluorescent sensor using this riboswitch fused to the RNA aptamer, Spinach2. When assayed anaerobically, the resulting sensor responds in vitro to Fe II , as well as to Mn II , Co II , Ni II , and Zn II , but only in the cases of Fe II and Mn II do the apparent K d values (0.4 and 11 μM, respectively) fall within the range of labile metal concentrations maintained by known metalloregulators. We also show that the sensor-which is, to the best of our knowledge, the first reversible genetically encoded fluorescent sensor for Fe II -responds to iron in Escherichia coli cells. Finally, we demonstrate that the putative metal exporters directly downstream of two czcD riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results indicate that iron merits consideration as a plausible physiological ligand for czcD riboswitches, although a response to general metal stress cannot be ruled out at present.

Translesion DNA synthesis (TLS) bypasses DNA lesions encountered during S-phase and is critical for cell survival after exposure to DNA-damaging agents. In humans, Rad6/Rad18 attaches single ubiquitin moieties (i.e., monoubiquitination) to proliferating cell nuclear antigen (PCNA) sliding clamps encircling primer/template (P/T) junctions that are stalled at DNA lesions. TLS occurs via PCNA monoubiquitination-independent and -dependent pathways, and both contribute to cell survival. The interaction of Rad6/Rad18 with PCNA is paramount to PCNA monoubiquitination and remains poorly defined. In particular, the location of the Rad6/Rad18 binding site on PCNA is unknown. Many PCNA-binding proteins, particularly DNA polymerases (pols), converge on PCNA encircling stalled P/T junctions in human cells, and all interact in a similar manner with the universal binding sites on PCNA. We reasoned the following: if Rad6/Rad18 utilizes the universal binding sites (or nearby sites), then PCNA monoubiquitination may be suppressed by pols involved in TLS. Results from quantitative studies reveal that (1) a Y-family pol (pol η) and a B-family pol (pol δ) critical to TLS each inhibit the transfer of ubiquitin from Rad6/Rad18 to PCNA and that (2) the observed inhibitions are dependent on the interaction of these pols with PCNA encircling DNA. These studies suggest that Rad6/Rad18 utilizes the universal PCNA-binding sites or nearby sites and, hence, competes for PCNA encircling DNA with pols η and δ and possibly other PCNA-binding proteins involved in TLS. These findings provide valuable insight into the nature of the interaction between Rad6/Rad18 and PCNA and have important implications for the division of human TLS pathways.

The number of self-cleaving ribozymes has increased sharply in recent years, giving rise to elaborations of the four known ribozyme catalytic strategies, α, β, γ, and δ. One such extension is utilized by the twister ribozyme, which is hypothesized to conduct δ, or general acid catalysis, via N3 of the syn adenine +1 nucleobase indirectly via buffer catalysis at biological pH and directly at lower pH. Herein, we test the δ catalysis role of A1 via chemical rescue and the catalytic relevance of the syn orientation of the nucleobase by conformational analysis. Using inhibited twister ribozyme variants with A1(N3) deaza or A1 abasic modifications, we observe >100-fold chemical rescue effects in the presence of protonatable biological small molecules such as imidazole and histidine, similar to observed rescue values previously reported for C75U/C76Δ in the HDV ribozyme. Brønsted plots for the twister variants support a model in which small molecules rescue catalytic activity via a proton transfer mechanism, suggesting that A1 in the wild type is involved in proton transfer, most likely general acid catalysis. Additionally, through glycosidic conformational analysis in an appropriate background that accommodates the bromine atom, we observe that an 8BrA1-modified twister ribozyme is up to 10-fold faster than a nonmodified A1 ribozyme, supporting crystallographic data that show that A1 is syn when conducting proton transfer. Overall, this study provides functional evidence that the nucleotide immediately downstream of the cleavage site participates directly or indirectly in general acid-base catalysis in the twister ribozyme while occupying the syn conformation.

As the influence of translation rates on protein folding and function has come to light, the mechanisms by which translation speed is modulated have become an important issue. One mechanism entails the generation of force by the nascent protein. Cotranslational processes, such as nascent protein folding, the emergence of unfolded nascent chain segments from the ribosome's exit tunnel, and insertion of the nascent chain into or translocation of the nascent chain through membranes, can generate forces that are transmitted back to the peptidyl transferase center and affect translation rates. In this Perspective, we examine the processes that generate these forces, the mechanisms of transmission along the ribosomal exit tunnel to the peptidyl transferase center, and the effects of force on the ribosome's catalytic cycle. We also discuss the physical models that have been developed to predict and explain force generation for individual processes and speculate about other processes that may generate forces that have yet to be tested.

In solution, proteins fluctuate among many conformational substates, with their relative free energies determining substate populations and energy barriers determining conformational exchange kinetics. It has been suggested that members of the conformational ensemble may be responsible for different protein functions, although it is generally difficult to test such a proposal in most systems. A model protein for deciphering individual substate contributions is the homodimeric Saccharomyces cerevisiae chorismate mutase (ScCM) enzyme, which is negatively and positively regulated by tyrosine and tryptophan, respectively. Previous X-ray crystallography structures revealed two equivalent allosteric binding pockets that can be occupied by either tryptophan or tyrosine. We proposed that under cellular conditions there are six potential states of ScCM: no allosteric effector bound, a single tyrosine bound, a single tryptophan bound, two tyrosines bound, two tryptophans bound, and a mixed bound state in which tyrosine and tryptophan occupy different allosteric sites. We used isothermal titration calorimetry and solution-state nuclear magnetic resonance spectroscopy to confirm the existence of all six states and construct the complete six-state equilibrium binding profile. We were also able to assign enzyme activities to each state, which allowed us to derive the enzyme activity landscape across the range of cellular concentrations of tyrosine and tryptophan. Surprisingly, the mixed bound state had the highest enzyme activity, which suggested that the shikimate pathway is shunted toward tyrosine production under most conditions.

RNAs are involved in myriad cellular events. In general, RNA function is affected by cellular conditions. For instance, molecular crowding promotes RNA folding through compaction of the RNA. Metabolites generally destabilize RNA secondary structure, which improves RNA folding cooperativity and increases the fraction of functional RNA. Our recent studies demonstrate that cellular concentrations of amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation. However, effects of other cellular magnesium ion chelators on RNA function have not been tested. Herein, we report that nucleotide diphosphate-chelated magnesium, which is of intermediate strength, promotes RNA catalysis much like aaCM. Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.50 mM Mg 2+ free . We tested the self-cleaving activity of the hammerhead ribozyme in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg 2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.

Thymidylate synthase (TS) is a dimeric enzyme conserved in all life forms that exhibits the allosteric feature of half-the-sites activity. Neither the reason for nor the mechanism of this phenomenon is understood. We used a combined nuclear magnetic resonance (NMR) and molecular dynamics approach to study a stable intermediate preceding hydride transfer, which is the rate-limiting and half-the-sites step. In NMR titrations with ligands leading to this intermediate, we measured chemical shifts of the apoenzyme (lig 0 ), the saturated holoenzyme (lig 2 ), and the typically elusive singly bound (lig 1 ) states. Approximately 40 amides showed quartet patterns providing direct NMR evidence of coupling between the active site and probes >30 Å away in the distal subunit. Quartet peak patterns have symmetrical character, indicating reciprocity in communicating the first and second binding events to the distal protomer. Quartets include key catalytic residues and map to the dimer interface β-sheet, which also represents the shortest path between the two active sites. Simulations corroborate the coupling observed in solution in that there is excellent overlap between quartet residues and main-chain atoms having intersubunit cross-correlated motions. Simulations identify five hot spot residues, three of which lie at the kink in the unique β-bulge abutting the active sites on either end of the sheet. Interstrand cross-correlated motions become more organized and pronounced as the enzyme progresses from lig 0 to lig 1 and ultimately lig 2 . Coupling in the apparently symmetrical complex has implications for half-the-sites reactivity and potentially resolves the paradox of inequivalent TS active sites despite the vast majority of X-ray structures appearing to be symmetrical.

Cfr is a radical S -adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 ( Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro . Here we show that Cfr homologues from Bacillus amyloliquefaciens , Enterococcus faecalis , Paenibacillus lautus , and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe 2+ ion in a rubredoxin-type Cys 4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe 2+ -binding site by Zn 2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.

Pyrroloquinoline quinone (PQQ) is an essential redox cofactor in bacterial calcium- and lanthanide-dependent alcohol dehydrogenases. Although certain bacteria are known to synthesize and secrete PQQ, little is known about trafficking of this cofactor within and between cells. Here, we show that a previously uncharacterized periplasmic (solute) binding protein from Methylobacterium extorquens AM1, here renamed PqqT, binds 1 equiv of PQQ with high affinity ( K d = 50 nM). UV-visible and spectrofluorometric titrations establish that PqqT binds an unhydrated form of PQQ with distinct spectral features from the cofactor in free solution. To our knowledge, PqqT is the first solute-binding protein identified for PQQ and the first protein implicated in cellular trafficking of the cofactor. We propose that PqqT, which is encoded adjacent to a putative ATP-binding cassette transporter in the M. extorquens genome, is involved in uptake of exogenous PQQ to supplement endogenous cofactor biosynthesis. These results support the emerging importance of PQQ transfer within microbial and microbe-host communities.

Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated. High-resolution crystal structures (<2 Å) under different illumination conditions with either FMN or 5dFMN chromophores reveal three conformations of the highly conserved glutamine 293. An allosteric hydrogen bond network linking the chromophore via Gln293 to the auxiliary A'α helix is observed. With FMN, a "flip" of the Gln293 side chain occurs between dark and lit states. 5dFMN cannot hydrogen bond through the C5 position and proved to be unable to support Au1a domain dimerization. Under blue light, the Gln293 side chain instead "swings" away in a conformation distal to the chromophore and not previously observed in existing LOV domain structures. Together, the multiple side chain conformations of Gln293 and functional analysis of 5dFMN provide new insight into the structural requirements for LOV domain activation.

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate β subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a Mn II 2 cluster by free superoxide to yield a metal-based Mn III Mn IV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive β subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.

Deoxynucleotide misincorporation efficiencies can span a wide 10 4 -fold range, from ∼10 -2 to ∼10 -6 , depending principally on polymerase (pol) identity and DNA sequence context. We have addressed DNA pol fidelity mechanisms from a transition-state (TS) perspective using our "tool-kit" of dATP- and dGTP-β,γ substrate analogues in which the pyrophosphate leaving group (p K a4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p K a4 values from 7.8 (CF 2 ) to 12.3 [C(CH 3 ) 2 ]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( k pol ) versus p K a4 , for Y-family error-prone pol η and X-family pols λ and β to determine the extent to which different electrostatic active site environments alter k pol values. The apparent chemical rate constant ( k pol ) is the rate-determining step for the three pols. The pols each exhibit a distinct catalytic signature that differs for formation of right (A·T) and wrong (G·T) incorporations observed as changes in slopes and displacements of the Brønsted lines, in relation to a reference LFER. Common to this signature among all three pols is a split linear pattern in which the analogues containing two halogens show k pol values that are systematically lower than would be predicted from their p K a4 values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p K a4 values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet)-dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an S N 2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with Ca II binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for Ln III s and Y III over Ca II . Here we present the nuclear magnetic resonance solution structure of LanM complexed with Y III . This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein's picomolar affinity for Ln III s, and it suggests a role of unusual N i+1 -H···N i hydrogen bonds, in which LanM's unique EF-hand proline residues are engaged, in selective Ln III recognition. This work sets the stage for a detailed mechanistic understanding of LanM's Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technologically important metals.

Enzymes show two distinct transport behaviors in the presence of their substrates in solution. First, their diffusivity enhances with an increasing substrate concentration. In addition, enzymes perform directional motion toward regions with a high substrate concentration, termed as chemotaxis. While a variety of enzymes has been shown to undergo chemotaxis, there remains a lack of quantitative understanding of the phenomenon. Here, we derive a general expression for the active movement of an enzyme in a concentration gradient of its substrate. The proposed model takes into account both the substrate-binding and catalytic turnover step, as well as the enhanced diffusion of the enzyme. We have experimentally measured the chemotaxis of a fast and a slow enzyme: urease under catalytic conditions and hexokinase for both full catalysis and for simple noncatalytic substrate binding. There is good agreement between the proposed model and the experiments. The model is general, has no adjustable parameters, and only requires three experimentally defined constants to quantify chemotaxis: enzyme-substrate binding affinity ( K d ), Michaelis-Menten constant ( K M ), and level of diffusion enhancement in the associated substrate (α).

Inherited mutations of transthyretin (TTR) destabilize its structure, leading to aggregation and familial amyloid disease. Although numerous crystal structures of wild-type (WT) and mutant TTRs have been determined, they have failed to yield a comprehensive structural explanation for destabilization by pathogenic mutations. To identify structural and dynamic variations that are not readily observed in the crystal structures, we used NMR to study WT TTR and three kinetically and/or thermodynamically destabilized pathogenic variants (V30M, L55P, and V122I). Sequence-corrected chemical shifts reveal important structural differences between WT and mutant TTR. The L55P mutation linked to aggressive early onset cardiomyopathy and polyneuropathy induces substantial structural perturbations in both the DAGH and CBEF β-sheets, whereas the V30M polyneuropathy-linked substitution perturbs primarily the CBEF sheet. In both variants, the structural perturbations propagate across the entire width of the β-sheets from the site of mutation. Structural changes caused by the V122I cardiomyopathy-associated mutation are restricted to the immediate vicinity of the mutation site, directly perturbing the subunit interfaces. NMR relaxation dispersion measurements show that WT TTR and the three pathogenic variants undergo millisecond time scale conformational fluctuations to populate a common excited state with an altered structure in the subunit interfaces. The excited state is most highly populated in L55P. The combined application of chemical shift analysis and relaxation dispersion to these pathogenic variants reveals differences in ground state structure and in the population of a transient excited state that potentially facilitates tetramer dissociation, providing new insights into the molecular mechanism by which mutations promote TTR amyloidosis.

There is considerable interest in how "second-shell" interactions between protein side chains and metal ligands might modulate Mn(II) ion redox properties and reactivity in metalloenzymes. One such Mn-dependent enzyme is oxalate decarboxylase (OxDC), which catalyzes the disproportionation of oxalate monoanion into formate and CO 2 . Electron paramagnetic resonance (EPR) studies have shown that a mononuclear Mn(III) ion is formed in OxDC during catalytic turnover and that the removal of a hydrogen bond between one of the metal ligands (Glu101) and a conserved, second-shell tryptophan residue (Trp132) gives rise to altered zero-field splitting parameters for the catalytically important Mn(II) ion. We now report heavy-atom kinetic isotope effect measurements on the W132F OxDC variant, which test the hypothesis that the Glu101/Trp132 hydrogen bond modulates the stability of the Mn(III) ion during catalytic turnover. Our results suggest that removing the Glu101/Trp132 hydrogen bond increases the energy of the oxalate radical intermediate from which decarboxylation takes place. This finding is consistent with a model in which the Glu101/Trp132 hydrogen bond in WT OxDC modulates the redox properties of the Mn(II) ion.

Despite purines making up one of the largest classes of metabolites in a cell, little is known about the regulatory mechanisms that facilitate efficient purine production. Under conditions resulting in high purine demand, enzymes within the de novo purine biosynthetic pathway cluster into multienzyme assemblies called purinosomes. Purinosome formation has been linked to molecular chaperones HSP70 and HSP90; however, the involvement of these molecular chaperones in purinosome formation remains largely unknown. Here, we present a new-found biochemical mechanism for the regulation of de novo purine biosynthetic enzymes mediated through HSP90. HSP90-client protein interaction assays were employed to identify two enzymes within the de novo purine biosynthetic pathway, PPAT and FGAMS, as client proteins of HSP90. Inhibition of HSP90 by STA9090 abrogated these interactions and resulted in a decrease in the level of available soluble client protein while having no significant effect on their interactions with HSP70. These findings provide a mechanism to explain the dependence of purinosome assembly on HSP90 activity. The combined efforts of molecular chaperones in the maturation of PPAT and FGAMS result in purinosome formation and are likely essential for enhancing the rate of purine production to meet intracellular purine demand.

A diverse set of organisms has adapted to live under extreme conditions. The molecular origin of the stability is unclear, however. It is not known whether the adaptation of functional RNAs, which have intricate tertiary structures, arises from strengthening of tertiary or secondary structure. Herein we evaluate effects of sequence changes on the thermostability of tRNA phe using experimental and computational approaches. To separate out effects of secondary and tertiary structure on thermostability, we modify base pairing strength in the acceptor stem, which does not participate in tertiary structure. In dilute solution conditions, strengthening secondary structure leads to non-two-state thermal denaturation curves and has small effects on thermostability, or the temperature at which tertiary structure and function are lost. In contrast, under cellular conditions with crowding and Mg 2+ -chelated amino acids, where two-state cooperative unfolding is maintained, strengthening secondary structure enhances thermostability. Investigation of stabilities of each tRNA stem across 44 organisms with a range of optimal growing temperatures revealed that organisms that grow in warmer environments have more stable stems. We also used Shannon entropies to identify positions of higher and lower information content, or sequence conservation, in tRNA phe and found that secondary structures have modest information content allowing them to drive thermal adaptation, while tertiary structures have maximal information content hindering them from participating in thermal adaptation. Base-paired regions with no tertiary structure and modest information content thus offer a facile evolutionary route to enhancing the thermostability of functional RNA by the simple molecular rules of base pairing.

This Perspective focuses on RNA in biological and nonbiological compartments resulting from liquid-liquid phase separation (LLPS), with an emphasis on origins of life. In extant cells, intracellular liquid condensates, many of which are rich in RNAs and intrinsically disordered proteins, provide spatial regulation of biomolecular interactions that can result in altered gene expression. Given the diversity of biogenic and abiogenic molecules that undergo LLPS, such membraneless compartments may have also played key roles in prebiotic chemistries relevant to the origins of life. The RNA World hypothesis posits that RNA may have served as both a genetic information carrier and a catalyst during the origin of life. Because of its polyanionic backbone, RNA can undergo LLPS by complex coacervation in the presence of polycations. Phase separation could provide a mechanism for concentrating monomers for RNA synthesis and selectively partition longer RNAs with enzymatic functions, thus driving prebiotic evolution. We introduce several types of LLPS that could lead to compartmentalization and discuss potential roles in template-mediated non-enzymatic polymerization of RNA and other related biomolecules, functions of ribozymes and aptamers, and benefits or penalties imparted by liquid demixing. We conclude that tiny liquid droplets may have concentrated precious biomolecules and acted as bioreactors in the RNA World.

The core of the loline family of insecticidal alkaloids is the bicyclic pyrrolizidine unit with an additional strained ether bridge between carbons 2 and 7. Previously reported genetic and in vivo biochemical analyses showed that the presumptive iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, LolO, is required for installation of the ether bridge upon the pathway intermediate, 1- exo-acetamidopyrrolizidine (AcAP). Here we show that LolO is, in fact, solely responsible for this biosynthetic four-electron oxidation. In sequential 2OG- and O 2 -consuming steps, LolO removes hydrogens from C2 and C7 of AcAP to form both carbon-oxygen bonds in N-acetylnorloline (NANL), the precursor to all other lolines. When supplied with substoichiometric 2OG, LolO only hydroxylates AcAP. At higher 2OG:AcAP ratios, the enzyme further processes the alcohol to the tricyclic NANL. Characterization of the alcohol intermediate by mass spectrometry and nuclear magnetic resonance spectroscopy shows that it is 2- endo-hydroxy-1- exo-acetamidopyrrolizidine (2- endo-OH-AcAP). Kinetic and spectroscopic analyses of reactions with site-specifically deuteriated AcAP substrates confirm that the C2-H bond is cleaved first and that the responsible intermediate is, as expected, an Fe IV -oxo (ferryl) complex. Analyses of the loline products from cultures fed with stereospecifically deuteriated AcAP precursors, proline and aspartic acid, establish that LolO removes the endo hydrogens from C2 and C7 and forms both new C-O bonds with retention of configuration. These findings delineate the pathway to an important class of natural insecticides and lay the foundation for mechanistic dissection of the chemically challenging oxacyclization reaction.

Charged bases occur in RNA enzymes, or ribozymes, where they play key roles in catalysis. Cationic bases donate protons and perform electrostatic catalysis, while anionic bases accept protons. We previously published simulations of rate-pH profiles for ribozymes in terms of species plots for the general acid and general base that have been useful for understanding how ribozymes respond to pH. In that study, we did not consider interaction between the general acid and general base or interaction with other species on the RNA. Since that report, diverse small ribozyme classes have been discovered, many of which have charged nucleobases or metal ions in the active site that can either directly interact and participate in catalysis or indirectly interact as "influencers". Herein, we simulate experimental rate-pH profiles in terms of species plots in which reverse protonated charged nucleobases interact. These analyses uncover two surprising features of pH-dependent enzyme kinetics. (1) Cooperativity between the general acid and general base enhances population of the functional forms of a ribozyme and manifests itself as hidden or "dark" pK a shifts, real pK a shifts that accelerate the reaction but are not readily observed by standard experimental approaches, and (2) influencers favorably shift the pK a s of proton-transferring nucleobases and manifest themselves as "wavy" rate-pH profiles. We identify parallels with the protein enzyme literature, including reverse protonation and wavelike behavior, while pointing out that RNA is more prone to reverse protonation. The complexities uncovered, which arise from simple pairwise interactions, should aid deconvolution of complex rate-pH profiles for RNA and protein enzymes and suggest veiled catalytic devices for promoting catalysis that can be tested by experiment and calculation.

RNA enzymes, or ribozymes, catalyze internal phosphodiester bond cleavage using diverse catalytic strategies. These include the four classic strategies: in-line nucleophilic attack, deprotonation of the 2'-OH nucleophile, protonation of the 5'-O leaving group, and stabilization of developing charge on the nonbridging oxygen atoms of the scissile phosphate. In addition, we recently identified two additional ribozyme strategies: acidification of the 2'-OH and release of the 2'-OH from inhibitory interactions. Herein, we report inverse thio effects in the presence of glmS ribozyme variants and a 1-deoxyglucosamine 6-phosphate cofactor analogue and demonstrate that activation of the 2'-OH nucleophile is promoted by competitive hydrogen bonding among diverse ribozyme moieties for the pro-R P nonbridging oxygen. We conclude that the glmS ribozyme uses an overdetermined set of competing hydrogen bond donors in its active site to ensure potent activation and regulation by the cofactor. Nucleophile activation through competitive, overdetermined hydrogen bonding could be a general strategy for ribozyme activation and may be applicable for controlling the function of ribozymes and riboswitches in the laboratory.