Search

The first step of histidine biosynthesis in Acinetobacter baumannii , the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N 1 -(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, is catalyzed by the hetero-octameric enzyme ATP phosphoribosyltransferase, a promising target for antibiotic design. The catalytic subunit, HisG S , is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in k cat . Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisG S ) and activated (ATPPRT) forms of the enzyme. A pH-rate profile showed that maximum catalysis is achieved above pH 8.0. Surprisingly, at 25 °C, k cat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisG S . The HisG S -catalyzed reaction is limited by the chemical step, as suggested by the enhancement of k cat when Mg 2+ was replaced by Mn 2+ , and by the lack of a pre-steady-state burst of product formation. Conversely, the ATPPRT-catalyzed reaction rate is determined by PRATP diffusion from the active site, as gleaned from a substantial solvent viscosity effect. A burst of product formation could be inferred from pre-steady-state kinetics, but the first turnover was too fast to be directly observed. Lowering the temperature to 5 °C allowed observation of the PRATP formation burst by ATPPRT. At this temperature, the single-turnover rate constant was significantly higher than k cat , providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.

The enzyme 2'-deoxynucleoside 5'-phosphate N -hydrolase 1 (DNPH1) catalyzes the N -ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum k cat / K M , two groups with an average p K a of 6.4 must be deprotonated, while two groups with an average p K a of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to k cat / K M . Solvent deuterium isotope effects on k cat / K M and k cat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

( R )-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to ( R )-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at -5, 0, and 5 °C show a much higher steady-state rate constant ( k cat ) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants ( k STO ) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on k cat ( D k cat ) and k STO ( D k STO ) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH D k cat and D k STO are generally temperature-independent. The D k STO data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 °C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at -5 °C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 °C, the His-to-Asn mutation in psychrophilic HBDH decreases D k cat to 3.1, suggesting a decrease in transition-state symmetry, while the His-to-Asn mutation in mesophilic HBDH increases D k cat to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis, being allosterically inhibited by the final product of the pathway. Allosteric inhibition of long-form ATPPRTs by histidine has been extensively studied, but inhibition of short-form ATPPRTs is poorly understood. Short-form ATPPRTs are hetero-octamers formed by four catalytic subunits (HisG S ) and four regulatory subunits (HisZ). HisG S alone is catalytically active and insensitive to histidine. HisZ enhances catalysis by HisG S in the absence of histidine but mediates allosteric inhibition in its presence. Here, steady-state and pre-steady-state kinetics establish that histidine is a noncompetitive inhibitor of short-form ATPPRT and that inhibition does not occur by dissociating HisG S from the hetero-octamer. The crystal structure of ATPPRT in complex with histidine and the substrate 5-phospho-α-d-ribosyl-1-pyrophosphate was determined, showing histidine bound solely to HisZ, with four histidine molecules per hetero-octamer. Histidine binding involves the repositioning of two HisZ loops. The histidine-binding loop moves closer to histidine to establish polar contacts. This leads to a hydrogen bond between its Tyr263 and His104 in the Asp101-Leu117 loop. The Asp101-Leu117 loop leads to the HisZ-HisG S interface, and in the absence of histidine, its motion prompts HisG S conformational changes responsible for catalytic activation. Following histidine binding, interaction with the histidine-binding loop may prevent the Asp101-Leu117 loop from efficiently sampling conformations conducive to catalytic activation. Tyr263Phe- Pa HisZ-containing Pa ATPPRT, however, is less susceptible though not insensitive to histidine inhibition, suggesting the Tyr263-His104 interaction may be relevant to yet not solely responsible for transmission of the allosteric signal.

Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.

The five-membered nitrogen plus heteroatom rings known as azolines or in their oxidized form as azoles are very common in natural products and drugs. The oxidation of thiazoline to thiazole in the cyanobactin class of natural products is one of the several important transformations that are known to alter the biological properties of the compound. The ordering of the various chemical reactions that occur during cyanobactin biosynthesis is not fully understood. The structure of the flavin-dependent enzyme responsible for the oxidation of multiple thiazolines reveals it contains an additional domain that in other enzymes recognizes linear peptides. We characterize the activity of the enzyme on two substrates: one with a peptide leader and one without. Kinetics and biophysics reveal that the leader on the substrate is not recognized by the enzyme. The enzyme is faster on either substrate than the macrocyclase or protease in vitro. The enzyme has a preferred order of oxidation of multiple thiazolines in the same linear peptide.

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisG S ) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg 2+ -dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N 1 -(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisG S is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisG S , but steady-state kinetics and 31 P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg 2+ by Mn 2+ enhances catalysis by HisG S but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an S N 2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn 2+ increasing HisG S reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisG S 's catalytic rate, but decreases the hetero-octamer's, indicating that chemistry and product release are rate-limiting for HisG S and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisG S . These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisG S , accelerating chemistry beyond the product release rate.

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising druglike molecules because of their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases.

Mammalian β-hexosaminidases have been shown to play essential roles in cellular physiology and health. These enzymes are responsible for the cleavage of the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) from cellular substrates. One of these β-hexosaminidases, hexosaminidase D (HexD), encoded by the HEXDC gene, has received little attention. No mechanistic studies have focused on the role of this unusual nucleocytoplasmically localized β-hexosaminidase, and its cellular function remains unknown. Using a series of kinetic and mechanistic investigations into HexD, we define the precise catalytic mechanism of this enzyme and establish the identities of key enzymic residues. The preparation of synthetic aryl N-acetylgalactosaminide substrates for HexD in combination with measurements of kinetic parameters for wild-type and mutant enzymes, linear free energy analyses of the enzyme-catalyzed hydrolysis of these substrates, evaluation of the reaction by nuclear magnetic resonance, and inhibition studies collectively reveal the detailed mechanism of action employed by HexD. HexD is a retaining glycosidase that operates using a substrate-assisted catalytic mechanism, has a preference for galactosaminide over glucosaminide substrates, and shows a pH optimum in its second-order rate constant at pH 6.5-7.0. The catalytically important residues are Asp148 and Glu149, with Glu149 serving as the general acid/base residue and Asp148 as the polarizing residue. HexD is inhibited by Gal-NAG-thiazoline (Ki = 420 nM). The fundamental insights gained from this study will aid in the development of potent and selective probes for HexD, which will serve as useful tools to improve our understanding of the physiological role played by this unusual enzyme.