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Nudix hydrolase family proteins hydrolyse toxic by-products of cellular metabolism such as mutagenic nucleoside triphosphates, sugar nucleotides and signalling molecules. We studied the substrate specificities of Nudix hydrolases encoded by rv3672c and rv3040c from Mycobacterium tuberculosis and their respective homologues, msmeg_6185 and msmeg_2327 from M. smegmatis. The rv3672c- and msmeg_6185-encoded proteins (Rv3672 and MSMEG_6185, respectively) showed CoA pyrophosphatase (CoAse) activity that converted acyl-CoA to adenosine-3',5'-diphosphate (3', 5'-ADP) and 4-acyl phosphopantetheine. The efficiencies of Rv3672 and MSMEG_6185 in hydrolysing CoA derivatives were found to be higher than those of the Rv3040 and MSMEG_2327 (encoded by rv3040c and msmeg_2327, respectively). Further, amongst the substrates tested, Rv3672 and MSMEG_6185 used CoA and oxidized CoA as the most preferred substrates. Use of the M. smegmatis model showed that the expression of msmeg_6185 occurs in the log and stationary phases but declines during the late stationary phase and becomes undetectable during hypoxia. The co-culture competition experiments performed between the wild-type and Delta msmeg_6185 strains of M. smegmatis in different carbon sources revealed that the presence of msmeg_6185 provided growth fitness advantage to M. smegmatis, irrespective of the carbon source, implicating its function in regulation for the optimal physiological levels of acyl-CoAs in the cell.

Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4′-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis’s phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium’s cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.

Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4’-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3’, 5’-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organisations. The universal distribution of FAALs suggests they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.

PLP-dependent enzymes are attractive biocatalysts due to their ability to perform complexity building transformations on amine-containing, readily available substrates from the chiral pool such as amino acids. Although many classes of PLP-dependent enzymes are routinely employed in synthesis, select families remain underutilized due to limitations in practical scalability. For example, some PLP-dependent enzymes act on prohibitively expensive substrates such as coenzyme A-bound substrates. To address this challenge, we have investigated the mechanism of action of coenzyme A in gating catalysis of one α-oxoamine synthase, SxtA AOS. Through investigation of the reactivity of SxtA AOS with panels of substrates and in the presence of various coenzyme A mimics as well as monitoring PLP absorbances and the behavior of SxtA AOS variants, we have determined that activity is gated through the binding of the eastern phosphodiester group by the residue Arg142 that is located in a tunnel that leads to the active site. To circumvent this gating mechanism, a synthetic CoA mimic additive was designed that allows for enhanced reactivity with non-CoA substrates. These findings outline a strategy for employing AOS enzymes without the need for cost-prohibitive coenzyme A substrates.

During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

Acyl carrier proteins (ACP)s transport intermediates through many primary and secondary metabolic pathways. Studying the effect of substrate identity on ACP structure has been hindered by the lability of the thioester bond that attaches acyl substrates to the 4’-phosphopantetheine cofactor of ACP. Here we show that an acyl acyl-carrier protein synthetase (AasS) can be used in real time to shift the hydrolysis equilibrium towards favoring acyl-ACP during solution NMR spectroscopy. Only 0.005 molar equivalents of AasS enables one week of stability to palmitoyl-AcpP from Escherichia coli. 2D NMR spectra enabled with this method revealed that the tethered palmitic acid perturbs nearly every secondary structural region of AcpP. This technique will allow previously unachievable structural studies of unstable acyl-ACP species, contributing to the understanding of these complex biosynthetic pathways.

At the center of many complex biosynthetic pathways, the acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides. Carrier proteins covalently tether their cargo via a thioester linkage to a phosphopantetheine cofactor. Due to the labile nature of this linkage, chemoenzymatic methods have been developed that involve replacement of the thioester with a more stable amide or ester bond. We explored the importance of the thioester bond to the structure of the carrier protein by using solution NMR spectroscopy and molecular dynamics simulations. Remarkably, the replacement of sulfur with other heteroatoms results in significant structural changes, thus suggesting more rigorous selections of isosteric substitutes is needed.

The mitochondrial acyl carrier protein (human ACPM, yeast Acp1) is an essential mitochondrial protein. Through binding of nascent acyl chains on the serine (S112)-bound 4′-phosphopantetheine (4′-PP) cofactor, ACPM is involved in mitochondrial fatty acid synthesis and lipoic acid biogenesis. Recently, yeast Acp1 was found to interact with several mitochondrial complexes, including the iron–sulfur (Fe–S) cluster biosynthesis and respiratory complexes, via the binding to LYRM proteins, a family of proteins involved in assembly/stability of complexes. Importantly, the interaction of LYRM proteins with Acp1 was shown to be essential in maintaining integrity of mitochondrial complexes. In human, recent structures show that ACPM binding to LYRM proteins involves acyl chains attached to the 4′-PP cofactor. Here, we performed an detailed characterization of the mitochondrial interactome of human ACPM by mass spectrometry (MS) and demonstrate the crucial role of the 4′-PP cofactor in most of ACPM interactions. Specifically, we show that ACPM interacts with endogenous Fe–S cluster complex components through binding of the LYRM protein ISD11/LYRM4. Using knockdown experiments, we further determine that ACPM is essential for the stability of mitochondrial respiratory complexes I, II and III, as well as the Fe–S cluster biosynthesis complex. Finally, using native MS and a top-down MS approach, we show that C14, C16 and C18 3-keto-acyl chains on ACPM are implicated in binding to ISD11 through analysis of the recombinant ACPM–ISD11 complex. Taken together, our data provide novel understanding of the role of 4′-PP- and long acyl chains-dependent interactions in human ACPM function.

The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4'-phosphopantetheine, 4'-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker-ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein-protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.

FIT2 is a protein important for ER lipid metabolism and lipid droplet formation, but its function has remained mysterious. Becuwe et al. show that FIT2 is an acyl coenzyme A diphosphatase, and this activity is crucial for ER homeostasis and cellular lipid storage., The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl–coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl–CoA to yield acyl 4′-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl–CoA levels as key to ER homeostasis.

International audience; One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4 0-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4 0-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases.

Aim And Objectives: Phytophthora infestans (Mont.) de Bary, the fungal pathogen causes late blight, which results in devastating economic loss among the Solanaceae. The bacillus lipopeptides show the antagonistic activity against the many plant pathogens, among bacillus lipopeptides reported as the antifungal gene. Hence, to understand the in silico antifungal activity, we have selected gene iturin A (AXN89987) produced by Bacillus spp to check the molecular dynamics study with the effector proteins of the P. infestanse. In this concern, known effector proteins of P. infestans were subjected to the protein-protein interaction followed by simulation. Material and Method: turin A gene was amplified using the soil bacterium Bacillus subtilis with gene-specific primers, cloned into pTZ 57R/T vector and confirmed by sequencing. To get better insights, the protein model was developed for Iturin A using Modeller 9.17, using PDB structure of ID 4MRT (Phosphopantetheine transferase Sfp) and 1QR0 (4'-phosphopantetheinyl moiety of coenzyme A) as a template, it shared the identity 72% and expected P-value: 3e-121, respectively. The model quality was assessed using ProSA and PROCHECK programs. Results: The potency of modelled protein against effector proteins of P. infestans were evaluated in silico using the HADDOCK server and the results showed the high affinity of towards the effector protein Host ATG8 (PDB-5L83). Finally, the simulation was performed to the docked conformation of with Host ATG8 to further understand the stability of the complex using the Desmond program. Conclusion: Altogether, the protein-protein interaction and simulation study propose a new methodology and to uncover possible antifungal activity of iturin A against effector proteins of P. infestans.

PKAN, CoPAN, MePAN, and PDH‐E2 deficiency share key phenotypic features but harbor defects in distinct metabolic processes. Selective damage to the globus pallidus occurs in these genetic neurodegenerative diseases, which arise from defects in CoA biosynthesis (PKAN, CoPAN), protein lipoylation (MePAN), and pyruvate dehydrogenase activity (PDH‐E2 deficiency). Overlap of their clinical features suggests a common molecular etiology, the identification of which is required to understand their pathophysiology and design treatment strategies. We provide evidence that CoA‐dependent activation of mitochondrial acyl carrier protein (mtACP) is a possible process linking these diseases through its effect on PDH activity. CoA is the source for the 4′‐phosphopantetheine moiety required for the posttranslational 4′‐phosphopantetheinylation needed to activate specific proteins. We show that impaired CoA homeostasis leads to decreased 4′‐phosphopantetheinylation of mtACP. This results in a decrease of the active form of mtACP, and in turn a decrease in lipoylation with reduced activity of lipoylated proteins, including PDH. Defects in the steps of a linked CoA‐mtACP‐PDH pathway cause similar phenotypic abnormalities. By chemically and genetically re‐activating PDH, these phenotypes can be rescued, suggesting possible treatment strategies for these diseases., PKAN, CoPAN, MePAN and PDH‐E2 deficiency are neurodegenerative diseases that damage a specific area of the brain and in which mutated genes encode enzymes that play a role in intermediary metabolism. Restoring PDH activity rescues abnormalities caused by CoA biosynthesis defects in disease models.

Fatty acyl-AMP ligases (FAAL) are enzymes that establish the crosstalk between fatty acid synthases and polyketide synthases or non-ribosomal peptide synthetases by activating newly synthesized fatty acids, shuttling them towards polyketide synthesis. These enzymes are unique as they have evolved a strict rejection mechanism for the coenzyme-A but can recognize and react with the phosphopantetheine of acyl-carrier proteins. A strategically placed insertion in the N-terminal domain and the rigidity of the hydrophobic anchor holding the insertion is at the heart of such a discrimination mechanism, the molecular details of which is yet to be clearly understood. The unique structural features of the insertion have been exploited to filter out FAALs from other members of the superfamily, in silico. Interestingly, several independent studies have characterized FAALs from different organisms such as Legionella, Myxococcus, Ralstonia, Pseudoalteromonas, Sorangium, etc. to name a few, laying emphasis on the usage of a FAAL-specific biochemistry in these organisms, particularly for the production of important bioactive molecules. These bioactive molecules help in improving the fitness of these systems to tide over the competition for nutritional resources in their local niche. Substrate specificity of these enzymes is another interesting aspect, which may hint at their potential roles in the cell. Various substrate-bound crystal structures have been used to identify the determinants of chain-length specificity and predict the preferences for different FAALs (both mycobacterial and non-mycobacterial). The fascinating details of the mechanistic variation of these enzymes and the molecular determinants that define the chain-length specificity have been discussed herewith.

TamA is the adenylating enzyme that selects and activates fatty acids for tambjamine biosynthesis., The marine bacterium Pseudoalteromonas tunicata produces the bipyrrole antibiotic tambjamine YP1. This natural product is built from common amino acid and fatty acid building blocks in a biosynthetic pathway that is encoded in the tam operon which contains 19 genes. The exact role that each of these Tam proteins plays in tambjamine biosynthesis is not known. Here, we provide evidence that TamA initiates the synthesis and controls the chain length of the essential tambjamine fatty amine tail. Sequence analysis suggests the unusual TamA is comprised of an N-terminal adenylation (ANL) domain fused to a C-terminal acyl carrier protein (ACP). Mass spectrometry analysis of recombinant TamA revealed the surprising presence of bound C11 and C12 acyl-adenylate intermediates. Acylation of the ACP domain was observed upon attachment of the phosphopantetheine (4′-PP) arm to the ACP. We also show that TamA can transfer fatty acids ranging in chain length from C6–C13 to an isolated ACP domain. Thus TamA bridges the gap between primary and secondary metabolism by linking fatty acid and pyrrole biosynthetic pathways.

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10A, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD=62±13nM, followed by the binding of two more equivalents of holo-ACPP with KD=1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with KD=2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.

ALDH1L1, a member of the aldehyde dehydrogenase superfamily of enzymes, catalyzes the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. The enzyme is a tetramer of identical subunits, with each subunit consisting of three functional domains that originated from unrelated genes. The N- and C-terminal domains are catalytic, while the intermediate domain transfers the reaction intermediate from the N- to the C-terminal domain. The intermediate domain is an acyl carrier protein, possessing the covalently attached 4′-phosphopantetheine (4-PP) prosthetic group. This prosthetic group is known to function as a swinging arm transferring intermediates between enzymes in complex biosynthetic reactions. Here we have applied computer modeling using available structures of the three functional domains of ALDH1L1 to evaluate the extent of flexibility within the full-length protein. This approach allowed us to define positions of the 4-PP arm within the two catalytic domains and to predict N-terminal:intermediate and intermediate:C-terminal domain interfaces. Our models further suggested high degree of flexibility within the full-length enzyme.

Acyl carrier proteins (ACPs) are central hubs in polyketide and fatty acid biosynthetic pathways, but the fast motions of the ACP's phosphopantetheine (Ppant) arm make its conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we report that converting the terminal thiol of Escherichia coli ACP's Ppant arm into a thiocyanate activates this site to form a selective cross-link with the active site cysteine of its partner ketoacyl synthase (FabF). The reaction releases a cyanide anion, which can be detected by infrared spectroscopy. This represents a practical and generalizable method for obtaining and visualizing ACP-protein complexes relevant to biocatalysis and will be valuable in future structural and engineering studies.

The β-hydroxylation of l-histidine is the first step in the biosynthesis of the imidazolone base of the antifungal drug nikkomycin. The cytochrome P450 (NikQ) hydroxylates the amino acid while it is appended via a phosphopantetheine linker to the non-ribosomal peptide synthetase (NRPS) NikP1. The latter enzyme is comprised of an MbtH and single adenylation and thiolation domains, a minimal composition that allows for detailed binding and kinetics studies using an intact and homogeneous NRPS substrate. Electron paramagnetic resonance studies confirm that a stable complex is formed with NikQ and NikP1 when the amino acid is tethered. Size exclusion chromatography is used to further refine the principal components that are required for this interaction. NikQ binds NikP1 in the fully charged state, but binding also occurs when NikP1 is lacking both the phosphopantetheine arm and appended amino acid. This demonstrates that the interaction is mainly guided by presentation of the thiolation domain interface, rather than the attached amino acid. Electrochemistry and transient kinetics have been used to probe the influence of l-His-NikP1 binding on catalysis by NikQ. Unlike many P450s, the binding of substrate fails to induce significant changes on the redox potential and autoxidation properties of NikQ and slows down the binding of dioxygen to the ferrous enzyme to initiate catalysis. Collectively, these studies demonstrate a complex interplay between the NRPS maturation process and the recruitment and regulation of an auxiliary tailoring enzyme required for natural product biosynthesis.

Nosiheptide (NOS) is a highly modified thiopeptide antibiotic that displays formidable in vitro activity against a variety of Gram-positive bacteria. In addition to a central hydroxypyridine ring, NOS contains several other modifications, including multiple thiazole rings, dehydro-amino acids, and a 3,4-dimethylindolic acid (DMIA) moiety. The DMIA moiety is required for NOS efficacy and is synthesized from l-tryptophan in a series of reactions that have not been fully elucidated. Herein, we describe the role of NosJ, the product of an unannotated gene in the biosynthetic operon for NOS, as an acyl carrier protein that delivers 3-methylindolic acid (MIA) to NosK. We also reassign the role of NosI as the enzyme responsible for catalyzing the ATP-dependent activation of MIA and MIA’s attachment to the phosphopantetheine moiety of NosJ. Lastly, NosK catalyzes the transfer of the MIA group from NosJ-MIA to a conserved serine residue (Ser102) on NosK. The X-ray crystal structure of NosK, solved to 2.3 Å resolution, reveals that the protein is an α/β-fold hydrolase. Ser102 interacts with Glu210 and His234 to form a catalytic triad located at the bottom of an open cleft that is large enough to accommodate the thiopeptide framework.

Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo-LipD is very similar to other four-helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo-LipD are more restricted than in apo-LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo-LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg2+ or Ca2+ can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo-LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS-ACP from A. friuliensis that would allow the acyl-CoA ligase to interact preferentially with the LipD instead of binding to the FAS-ACP.

The natural substrates of the enzymes involved in type-II fatty acid biosynthesis (FAS-II) are acylated acyl carrier proteins (acyl-ACPs). The state of the art method to produce acyl-ACPs involves the transfer of a phosphopantetheine moiety from CoA to apo-ACP by E. coli holo-ACP synthase (EcACPS), yielding holo-ACP which subsequently becomes thioesterified with free fatty acids by the E. coli acyl-ACP synthase (EcAAS). Alternatively, acyl-ACPs can be synthesized by direct transfer of acylated phosphopantetheine moieties from acyl-CoA to apo-ACP by means of EcACPS. The need for native substrates to characterize the FAS-II enzymes of P. falciparum prompted us to investigate the potential and limit of the two methods to efficiently acylate P. falciparum ACP (PfACP) with respect to chain length and β-modification and in preparative amounts. The EcAAS activity is found to be independent from the oxidation state at the β-position and accepts fatty acids as substrates with chain lengths starting from C8 to C20, whereas EcACPS accepts very efficiently acyl-CoAs with chain lengths up to C16, and with decreasing activity also longer chains (C18 to C20). Methods were developed to synthesize and purify preparative amounts of high quality natural substrates that are fully functional for the enzymes of the P. falciparum FAS-II system., Graphical abstract fx1, Highlights • The apo-form of P. falciparum ACP (PfACP) has been purified to homogeneity. • PfACP can be acylated very efficiently and in preparative amounts using the improved EcACPS and EcAAS methods. • Small and long chain fatty acids can be transferred. • The acylation reaction is independent of the oxidation state at the β-position of the acyl-chains. • Acyl-PfACPs are fully functional substrates of the corresponding P. falciparum FAS-II enzymes.

Pantothenate kinase‐associated neurodegeneration (PKAN) is an inborn error of CoA metabolism causing dystonia, parkinsonism, and brain iron accumulation. Lack of a good mammalian model has impeded studies of pathogenesis and development of rational therapeutics. We took a new approach to investigating an existing mouse mutant of Pank2 and found that isolating the disease‐vulnerable brain revealed regional perturbations in CoA metabolism, iron homeostasis, and dopamine metabolism and functional defects in complex I and pyruvate dehydrogenase. Feeding mice a CoA pathway intermediate, 4′‐phosphopantetheine, normalized levels of the CoA‐, iron‐, and dopamine‐related biomarkers as well as activities of mitochondrial enzymes. Human cell changes also were recovered by 4′‐phosphopantetheine. We can mechanistically link a defect in CoA metabolism to these secondary effects via the activation of mitochondrial acyl carrier protein, which is essential to oxidative phosphorylation, iron–sulfur cluster biogenesis, and mitochondrial fatty acid synthesis. We demonstrate the fidelity of our model in recapitulating features of the human disease. Moreover, we identify pharmacodynamic biomarkers, provide insights into disease pathogenesis, and offer evidence for 4′‐phosphopantetheine as a candidate therapeutic for PKAN., Mutations in PANK2 cause pantothenate kinase‐associated neurodegeneration (PKAN), a neurodegeneration with brain iron accumulation (NBIA) disorder. This study presents a mouse model that recapitulates key features of the human disease and shows rescue by a coenzyme A pathway intermediate.

Phosphopantetheine adenylyl transferase catalyzes a rate limiting penultimate step of the multistep reaction which produces coenzyme A (CoA) as a final product. CoA is required as an essential cofactor in a number of metabolic reactions. Therefore inhibiting the function of this enzyme will lead to cell death in bacteria. Acinetobacter baumannii is multi drug resistant pathogen and causes infections in immunocompromised patients. AbPPAT has been cloned, expressed, purified and crystallized and structures of two complexes of AbPPAT with dephospho coenzyme A (dPCoA) and coenzyme A (CoA) have been determined. Both dPCoA and CoA molecules are observed in the substrate binding site of AbPPAT. A comparison with the structures of the complexes of PPAT from other species shows that the orientations of dPCoA are identical in all the structures. On the other hand, as observed from the structures of the complexes of CoA with PPAT, the orientations of CoA are found to differ considerably. This shows that the substrates occupy identical positions in the substrate binding sites of enzymes whereas the positions of inhibitors may differ. The binding studies carried out using fluorescence method and surface plasmon resonance techniques showed that binding affinity of CoA towards AbPPAT is nearly three times higher than that of dPCoA.

INTRODUCTION Mycobacterium tuberculosis (Mtb) is the leading global cause of lethal infection in humans and accounts for the largest number of drug-resistant infections by a single bacterial pathogen. Resistance is particularly high against the most widely prescribed tuberculosis (TB) drug, isoniazid. Isoniazid blocks synthesis of mycolates, ultralong-chain fatty acids that provide structure to the waxy coat that surrounds Mtb cells and are incorporated into some of its virulence lipids. There is currently no known method to block the synthesis of both mycolates and nonmycolate-containing virulence lipids of Mtb at a single point of control. One such control point is phosphopantetheinyl transferase (PptT). PptT transfers 4′-phosphopantetheine (Ppt) from coenzyme A (CoA) to acyl carrier proteins (ACPs) that synthesize the lipids critical to Mtb structural integrity and virulence. RATIONALE TB drug discovery often begins with whole-cell, high-throughput screens that yield compounds that kill Mtb by unknown means. Selection of Mtb mutants resistant to these compounds can indicate candidate targets of the active compound, but experimental validation is required to confirm the functionally relevant target, which is often an enzyme. A suitable target must be essential in vivo, such that its inhibition precludes development of TB in animal models, but also “vulnerable,” meaning that a pharmacologically attainable level of inhibition should be lethal to Mtb within a patient. The inhibitor should act only on Mtb, and resistance should be rare. RESULTS Screening a chemical library revealed an amidino-urea compound called “8918” that kills Mtb, including drug-resistant clinical isolates. 8918 inhibits Mtb in mice and spares other bacteria, yeast, and mammalian cells. Rare Mtb mutants resistant to 8918 bore a point mutation in the PptT gene rv2794c, altering an amino acid residue overlying the Ppt-binding pocket of PptT. When Mtb carried the mutant allele as an extra copy of rv2794c, the Mtb was protected from 8918. 8918 inhibited recombinant PptT, albeit noncompetitively and incompletely. The impact of 8918 on the Mtb metabolome and lipids was consistent with inhibition of PptT in the intact cell. A crystal structure of the PptT-8918 complex at 1.8-Å resolution confirmed that 8918 binds within the Ppt binding pocket, adjacent to the phosphoadenosine phosphate portion of CoA. Intact CoA remained in the PptT-8918 complex, but the Ppt arm was displaced, decreasing but not abolishing PptT’s catalytic activity. Strains of Mtb producing reduced amounts of PptT became hypersensitive to 8918. It was puzzling that even partial inhibition of PptT killed Mtb. We observed that mutants with disruption of rv2795c, a gene encoding a hypothetical protein, were also highly resistant to 8918. Recombinant Rv2795c protein hydrolyzed Ppt from a mycolate-building holo-ACP that is a substrate for PptT. The action of this Ppt hydrolase (PptH) resembled that of nonhomologous enzymes called ACP hydrolases that remove Ppt from ACPs in vitro but whose physiological function is unknown. CONCLUSION We identified a small molecule that kills Mtb by inhibiting PptT, demonstrating that a key enzyme in CoA metabolism is a viable target for TB drug development. Even partial inhibition of PptT is toxic to Mtb, likely because PptH synergizes with the inhibitor by undoing the PptT reaction. PptT and PptH are co-regulated by translation from the same operon, and thus Mtb cannot respond to inhibition of PptT by making more PptT without also generating more PptH. The joint functioning of PptT and PptH suggests that Mtb closely regulates the activation of ACPs. The transcriptional co-regulation and constitutive function of both members of the PptT-PptH couple suggests that a posttranslational signal that impairs PptT more than PptH could allow Mtb to rapidly reverse a prior commitment to synthesis of its metabolically most costly lipids.

In Mycobacterium tuberculosis, acyl carrier protein (AcpM)-mediated fatty acid synthase type II is integral for the synthesis of mycolic acids. AcpM, designated as an atypical ACP, comprises of a putative 33 amino acid long C-terminal extension which is distinctive in nature. Here, we aimed at devising an 'easy-to-go' method for the generation of crypto-AcpM loaded with a solvatochromic probe 7-Nitrobenz-2-oxa-1,3-diazol-4-yl, which is linked to the 4'-phosphopantetheine (Ppant) prosthetic group of AcpM. The crypto-AcpM, coupled with fluorescence spectroscopy and molecular dynamics simulation studies, was employed to explore the elusive dynamics of Ppant arm in AcpM. This investigation establishes the role of the flexible C-terminal extension of AcpM in regulating the prosthetic group sequestration ability by modulating the 'Asp-Ser-Leu' motif.

Type II polyketide synthases (PKSs) are protein assemblies, encoded by biosynthetic gene clusters in microorganisms, that manufacture structurally complex and pharmacologically relevant molecules. Acyl carrier proteins (ACPs) play a central role in biosynthesis by shuttling malonyl-based building blocks and polyketide intermediates to catalytic partners for chemical transformations. Because ACPs serve as central hubs in type II PKSs, they can also represent roadblocks to successfully engineering synthases capable of manufacturing ‘unnatural natural products.’ Therefore, understanding ACP conformational dynamics and protein interactions is essential to enable the strategic redesign of type II PKSs. However, the inherent flexibility and transience of ACP interactions pose challenges to gaining insight into ACP structure and function. In this review, we summarize how the application of chemical probes and molecular dynamic simulations has increased our understanding of the structure and function of type II PKS ACPs. We also share how integrating these advances in type II PKS ACP research with newfound access to key enzyme partners, such as the ketosynthase-chain length factor, sets the stage to unlock new biosynthetic potential.

Phosphopantetheinyl transferases (PPTases) are key elements in the modular syntheses performed by multienzyme systems such as polyketide synthases. PPTases transfer phosphopantetheine derivatives from Coenzyme A to carrier proteins (CPs), thus orchestrating substrate supply. We describe an efficient mass spectrometry‐based protocol for determining CP specificity for a particular PPTase in organisms possessing several candidate PPTases. We show that the CPs MbtL and PpsC, both involved in synthesis of essential metabolites in Mycobacterium tuberculosis, are exclusively activated by the type 2 PPTase PptT and not the type 1 AcpS. The assay also enables conclusive identification of the reactive serine on each CP.

Thioesters play essential roles in many biosynthetic pathways to fatty acids, esters, polyketides, and non-ribosomal peptides. Coenzyme A (CoA) and related phosphopantetheine thioesters are typically employed as activated acyl units for diverse C-C, C-O, and C-N coupling reactions. To study and control these enzymatic assembly lines in vitro and in vivo structurally simplified analogs such as N-acetylcysteamine (NAC) thioesters have been developed. This review gives an overview on experimental strategies enabled by synthetic NAC thioesters, such as the elucidation of complex biosynthetic pathways and enzyme mechanisms as well as precursor-directed biosynthesis and mutasynthesis. The review also summarizes synthetic protocols and protection group strategies to access these versatile synthetic tools, which are reactive and often unstable compounds. In addition, alternative phosphopantetheine thioester mimics are presented that can be used as protein tags or suicide inhibitors for protein crosslinking and off-loading probes to elucidate polyketide intermediates.

Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

The glycopeptide antibiotics are important clinical antibiotics that are currently employed against serious Gram-positive bacterial infections. Their chemical complexity means that their production is reliant upon the natural biosynthesis pathway, of which the key steps are the synthesis and side chain cyclisation of the peptide backbone. These processes rely upon a non-ribosomal peptide synthetase (NRPS) and several Cytochrome P450 enzymes that interact with the NRPS-bound peptide to generate the active aglycone. These P450 catalysts, known as Oxy enzymes, are crucial for the biosynthesis and yet the nature of their interactions with substrate peptides remains poorly understood. In this work, we have developed a range of synthetic inhibitor peptide substrates conjugated to an extended phosphopantetheine linker mimic and apply these to the characterisation of the first P450 from vancomycin biosynthesis, OxyBvan. These compounds display improved solubility and binding properties in comparison to isolated GPA precursor peptides and provide insights into the binding of such peptides to the P450 active site. These probes are applicable to the characterisation of other P450s from related biosynthetic machineries and are promising candidates for co-crystallisation with the Oxy enzymes.

Phosphopantetheine is a key structural element in biological acyl transfer reactions found embedded within coenzyme A (CoA). Phosphopantothenoylcysteine synthetase (PPCS) is responsible for installing a cysteamine group within phosphopantetheine. Therefore, it holds considerable potential as a drug target for developing new antimicrobials. In this study, we adapted a biochemical assay specific for bacterial PPCS to screen for inhibitors of CoA biosynthesis against a library of marine microbial derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces blancoensis led to the isolation of novel antibiotics (10-12, and 14) from the adipostatin class of molecules. The most potent molecule (10) displayed in vitro activity with IC50 = 0.93 µM, against S. pneumoniae PPCS. The whole cell antimicrobial assay against isolated molecules demonstrated their ability to penetrate bacterial cells and inhibit clinically relevant pathogenic strains. This establishes the validity of PPCS as a pertinent drug target, and the value of NPEs to provide new antibiotics. International Cooperative Biodiversity Groups/[U01 TW007404]/ICBG/Estados Unidos Hans W. Vahlteich Professorship/[NIH R35 GM118101]//Estados Unidos University of Michigan/[5T32GM008597]//Estados Unidos Rackham Merit Fellowship/[]/RMF/Estados Unidos UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA) UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Química

Biosynthetic enzyme complexes selectively catalyze challenging chemical transformations, including alkane functionalization (e.g., halogenation of threonine, Thr, by non-heme iron SyrB2). However, the role of complex formation in enabling reactivity and guiding selectivity is poorly understood, owing to the challenges associated with obtaining detailed structural information of the dynamically associating protein complexes. Combining over 10 ms of classical molecular dynamics of SyrB2 and the acyl carrier protein SyrB1 with large-scale QM/MM simulation, we investigate the substrate–protein and protein–protein dynamics that give rise to experimentally observed substrate positioning and reactivity trends. We confirm the presence of a hypothesized substrate-delivery channel in SyrB2 through free energy simulations that show channel opening with a low free energy barrier. We identify stabilizing interactions at the SyrB2/SyrB1 interface that are compatible with phosphopantatheine (PPant) delivery of substrate to SyrB2. By sampling metal–substrate distances observed in experimental spectroscopy of native SyrB2/SyrB1-PPant-S-Thr and non-native substrates, we characterize essential protein–substrate interactions that are responsible for substrate positioning, and thus, reactivity. We observe the hydroxyl sidechain and terminal amine of the native Thr substrate to form cooperative hydrogen bonds with a single N123 residue in SyrB2. In comparison, non-native substrates that lack the hydroxyl interact more flexibly with the protein and therefore can orient closer to the Fe center, explaining their preferential hydroxylation and higher turnover frequencies.

Fatty acid synthase 1 (FAS I) from Mycobacterium. tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4’-Phosphopantetheine (4’-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4’-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid.We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structurefunction analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Acyl carrier proteins (ACPs) play crucial roles in the biosynthesis of fatty acids, non-ribosomal polypeptides and polyketides. The three-dimensional NMR structure of Leishmania major holo-LmACP, belonging to the type II pathway, has been reported previously, but the structure of its apo-form and its conformational differences with the holo-form remain to be explored. Here we report the crystal structures of apo-LmACP (wild-type and S37A mutant) at 2.0 A resolution and compare their key features with the structures of holo-LmACP (wild-type) and other type II ACPs from Escherichia coli and Plasmodium falciparum. The crystal structure of apo-LmACP, which is homologous to other type II ACPs, displays some key structural rearrangements as compared to its holo-structure. Contrary to holo-form, which exists predominantly as a monomer, the apo-form exists as a mixture of monomeric and dimeric population in solution. In contrast to the closed structure of apo-LmACP, holo-LmACP structure was observed in an open conformation as a result of reorganization of specific helices and loops. We propose that the structural changes exhibited by LmACP occur due to the attachment of the phosphopantetheine arm and may be a prerequisite for the initiation of fatty acid synthesis. The movement of helix 3 may also play a role in the dissociation of holo-LmACP from its cognate enzymes of the FAS II pathway.

Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy generation, contributing to the proton motive force used to produce ATP. It couples the transfer of two electrons between NADH and quinone to translocation of four protons across the membrane. It is the largest protein assembly of bacterial and mitochondrial respiratory chains, composed, in mammals, of up to 45 subunits with a total molecular weight of ∼1 MDa. Bacterial enzyme is about half the size, providing the important “minimal” model of complex I. The l-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. Previously, we have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus and of the membrane domain from Escherichia coli, followed by the atomic structure of intact, entire complex I from T. thermophilus. Recently, we have solved by cryo-EM a first complete atomic structure of mammalian (ovine) mitochondrial complex I. Core subunits are well conserved from the bacterial version, whilst supernumerary subunits form an interlinked, stabilizing shell around the core. Subunits containing additional cofactors, including Zn ion, NADPH and phosphopantetheine, probably have regulatory roles. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The structure of mammalian enzyme provides many insights into complex I mechanism, assembly, maturation and dysfunction, allowing detailed molecular analysis of disease-causing mutations.

Nonribosomal peptide synthetases are large, multi-domain enzymes that produce peptide molecules with important biological activity such as antibiotic, antiviral, anti-tumor, siderophore and immunosuppressant action. The adenylation (A) domain catalyzes two reactions in the biosynthetic pathway. In the first reaction, it activates the substrate amino acid by adenylation and in the second reaction it transfers the amino acid onto the phosphopantetheine arm of the adjacent peptide carrier protein (PCP) domain. The conformation of the A domain differs significantly depending on which of these two reactions it is catalyzing. Recently, several structures of A-PCP di-domains have been solved using mechanism-based inhibitors to trap the PCP domain in the A domain active site. Here, we present an alternative strategy to stall the A-PCP di-domain, by engineering a disulfide bond between the native amino acid substrate and the A domain. Size exclusion studies showed a significant shift in apparent size when the mutant A-PCP was provided with cross-linking reagents, and this shift was reversible in the presence of high concentrations of reducing agent. The cross-linked protein crystallized readily in several of the conditions screened and the best crystals diffracted to ≈8 Å.

Phosphopantetheinylation is an essential post-translational protein modification to primary and secondary metabolic pathways that ensures bacterial cell viability and virulence, and it is used in the production of many pharmaceuticals. Traditional methods have not provided a comprehensive understanding of these modifications. By using chemical proteomic probes for adenylation and thiolation domains in nonribosomal peptide synthetases (NRPSs), chemoproteomics has been applied to survey and validate the cellular activity of 4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), which is a potent and selective small-molecule 4'-phosphopantetheinyl transferase (PPTase) inhibitor that attenuates secondary metabolism and viability of bacterial cells. ML267 inhibited Sfp-type PPTase and antagonized phosphopantetheinylation in cells, which resulted in a decrease in phosphopantetheinylated NRPSs and the attenuation of Sfp-PPTase-dependent metabolite production. These results indicate that this chemoproteomics platform should enable a precise interpretation of the cellular activities of Sfp-type PPTase inhibitors.

The mitochondrial acyl carrier protein (ACPM/NDUFAB1) is a central element of the mitochondrial fatty acid synthesis type II machinery. Originally ACPM was detected as a subunit of respiratory complex I but the reason for the association with the large enzyme complex remained elusive. Complex I from the aerobic yeast Yarrowia lipolytica comprises two different ACPMs, ACPM1 and ACPM2. They are anchored to the protein complex by LYR (leucine-tyrosine-arginine) motif containing protein (LYRM) subunits LYRM3 (NDUFB9) and LYRM6 (NDUFA6). The ACPM1-LYRM6 and ACPM2-LYRM3 modules are essential for complex I activity and assembly/stability, respectively. We show that in addition to the complex I bound fraction, ACPM1 is present as a free matrix protein and in complex with the soluble LYRM4(ISD11)/NFS1 complex implicated in Fe-S cluster biogenesis. We show that the presence of a long acyl chain bound to the phosphopantetheine cofactor is important for docking ACPMs to protein complexes and we propose that association of ACPMs and LYRMs is universally based on a new protein-protein interaction motif.

4'-Phosphopantetheinyl transferases (PPTase) post-translationally modify carrier proteins with a phosphopantetheine moiety, an essential reaction in all three domains of life. In the bacterial genus Mycobacteria, the Sfp-type PPTase activates pathways necessary for the biosynthesis of cell wall components and small molecule virulence factors. We solved the X-ray crystal structures and biochemically characterized the Sfp-type PPTases from two of the most prevalent Mycobacterial pathogens, PptT of M. tuberculosis and MuPPT of M. ulcerans. Structural analyses reveal significant differences in cofactor binding and active site composition when compared to previously characterized Sfp-type PPTases. Functional analyses including the efficacy of Sfp-type PPTase-specific inhibitors also suggest that the Mycobacterial Sfp-type PPTases can serve as therapeutic targets against Mycobacterial infections.

ACPs (acyl carrier proteins) play essential roles in the synthesis of fatty acids, polyketides and non-ribosomal polypeptides. ACP function requires the modification of the protein by attachment of 4′-phosphopantetheine to a conserved serine residue. The phosphopantetheine thiol acts to tether the starting materials and intermediates as their thioesters. ACPs are small highly soluble proteins composed of four α-helices. The helices form a bundle that acts as a hydrophobic sleeve that sequesters the acyl chains and activated thioesters from solvent. However, in the synthesis of fatty acids and complex lipids the enzymes of the pathway must access the thioester and the proximal carbon atoms in order to perform the needed chemistry. How such access is provided without exposure of the acyl chains to solvent has been a longstanding question due to the lack of acyl-ACP–enzyme complexes, a situation generally attributed to the brevity of the interactions of acyl-ACPs with their cognate enzymes. As discussed in the present review the access question has now been answered by four recent crystal structures, each of which shows that the entire acyl chain plus the 4′-phosphopantetheine prosthetic group partitions from the ACP hydrophobic sleeve into a hydrophobic pocket or groove of the enzyme protein, a process termed chain flipping.

Mammalian ACSF4-U26 (Acyl-CoA Synthetase Family Member 4), a protein of unknown function, comprises a putative adenylation-domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 shares more identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26 and prediction of its specificity suggests that it also serves to activate β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was alkali labile, suggesting a (thio)ester, and was not formed in a point-mutant devoid of the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was nearly specific for β-alanine, for which a KM of ≈ 5 µM was computed. The loaded enzyme was used to study the formation of a potential end-product. Among the twenty standard amino acids, only cysteine was able to cause unloading of the enzyme. This effect was mimicked by cysteamine and by dithiothreitol, and was unaffected by the absence of the PQQDH-related domain, suggesting that the β-alanine transfer onto thiols is catalyzed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification. This article is protected by copyright. All rights reserved.

Evaluation of new acyl carrier protein hydrolase (AcpH, EC 3.1.4.14) homologs from proteobacteria and cyanobacteria reveals significant variation in substrate selectivity and kinetic parameters for phosphopantetheine hydrolysis from carrier proteins. Evaluation with carrier proteins from both primary and secondary metabolic pathways reveals an overall preference for acyl carrier protein (ACP) substrates from type II fatty acid synthases, as well as variable activity for polyketide synthase ACPs and peptidyl carrier proteins (PCP) from non-ribosomal peptide synthases. We also demonstrate the kinetic parameters of these homologs for AcpP and the 11-mer peptide substrate YbbR. These findings enable the fully reversible labeling of all three classes of natural product synthase carrier proteins as well as full and minimal fusion protein constructs.

Tuberculosis Tuberculosis is a global health crisis that threatens to become worse as resistance to existing drugs emerges. Identifying ideal targets for drug development requires knowledge of weak points in biochemical pathways that are specific for the pathogen but are absent in hosts. Ballinger et al. identified a small molecule that inhibits the enzyme phosphopantetheinyl transferase (PptT), which is crucial for biosynthesis of mycobacterial structural and virulence lipids (see the Perspective by Mizrahi and Warner). Treatment resulted in selective killing of the bacteria in vitro and in a mouse model. The target pathways were made sensitive to PptT inhibition by a second enzyme, phosphopantetheine hydrolase, whose activity opposes that of the transferase. Science , this issue p. [eaau8959][1]; see also p. [457][2] [1]: /lookup/doi/10.1126/science.aau8959 [2]: /lookup/doi/10.1126/science.aaw5224

Carbon feedstocks from fossilized sources are being rapidly depleted due to rising demand for industrial and commercial applications. Many petroleum-derived chemicals can be directly or functionally substituted with chemicals derived from renewable feedstocks. Several short chain organic acids may fulfill this role using their functional groups as a target for chemical catalysis. Saccharomyces cerevisiae was engineered to produce short chain carboxylic acids (C6 to C10 ) from glucose using the heterologous Homo sapiens type I fatty acid synthase (hFAS). This synthase was activated by phosphopantetheine transfereases AcpS and Sfp from Escherichia coli and Bacillus subtilis, respectively, both in vitro and in vivo. hFAS was produced in the holo-form and produced carboxylic acids in vitro, confirmed by NADPH and ADIFAB assays. Overexpression of hFAS in a yeast FAS2 knockout strain, deficient in de novo fatty acid synthesis, demonstrated the full functional replacement of the native fungal FAS by hFAS. Two active heterologous short chain thioesterases (TEs) from Cuphea palustris (CpFatB1) and Rattus norvegicus (TEII) were evaluated for short chain fatty acid (SCFA) synthesis in vitro and in vivo. Three hFAS mutants were constructed: a mutant deficient in the native TE domain, a mutant with a linked CpFatB1 TE and a mutant with a linked TEII TE. Using the native yeast fatty acid synthase for growth, the overexpression of the hFAS mutants and the short-chain TEs (linked or plasmid-based) increased in vivo caprylic acid and total SCFA production up to 64-fold (63 mg/L) and 52-fold (68 mg/L), respectively, over the native yeast levels. Combined over-expression of the phosphopantetheine transferase with the hFAS mutant resulted in C8 titers of up to 82 mg/L and total SCFA titers of up to 111 mg/L.

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4' phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (-.164 kcal/mol) and B (-.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: -5.872 kcal/mol and B form: -5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.

The termination module of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) offloads the final product as an acid (occasionally also accompanied by cyclization) upon hydrolysis by employing thioesterase domains (TE-domains). Reductase domains (R-domains) of short-chain dehydrogenase/reductase (SDR) family offer an alternative offloading mechanism by reducing 4'-phosphopantetheine (4'-PPant) arm-tethered peptidyl chain, a thioester, to an aldehyde or an alcohol. Recent studies have highlighted their functional importance, for instance in the glycopeptidolipid (GPL) biosynthesis of Mycobacterium smegmatis, where the resulting alcoholic group is the site for subsequent modifications such as glycosylations. The mechanistic understanding of how these R-domains function in the context of multi-modular NRPS and PKS is poorly understood. In this study, conformational differences in functionally important loops, not reported previously, were identified in a new crystal form of R-domain which may be relevant to functioning in the context of assembly-line NRPS and PKS enzymology. Here, we propose a concerted loop movement model that allows gating of cofactor binding to these enzymes, enabling the release of the final product only after the substrate has reached the active site during biosynthesis, and therefore distinct from a canonical single domain SDR family of enzymes.

International audience; We have developed a chemoenzymatic route to coenzyme A (CoASH) and its disulfide that is amenable to gram-scale synthesis using standard laboratory equipment. By synthesizing the symmetrical disulfide of pantetheine (pantethine), we avoided the need to mask the reactive sulfhydryl and also prevented sulfur oxidation byproducts. No chromatography is required in our synthetic route to pantethine, which facilitates scale-up. Furthermore, we discovered that all three enzymes of the CoASH salvage pathway (pantetheine kinase, phosphopantetheine adenyltransferase, and dephospho-coenzyme A kinase) accept the disulfide of the natural substrates and functionalize both ends of the molecules. This yields CoA disulfide as the product of the enzymatic cascade, a much more stable form of the cofactor. Free CoASH can be prepared by in situ S–S reduction.

The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein, we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA libraries using a wide variety of phylogenetically diverse bacterial hosts.