A Type 1 Aldolase, NahE, Catalyzes a Stereoselective Nitro-Michael Reaction: Synthesis of ß-Aryl-γ-nitrobutyric Acids.

Michael addition reactions are highly useful in organic synthesis and are commonly accomplished using organocatalysts. However, the corresponding biocatalytic Michael additions are rare, typically lack synthetically useful substrate scope, and suffer from low stereoselectivity. Herein we report a biocatalytic nitro-Michael addition, catalyzed by NahE, that proceeds with low catalyst loading at room temperature in moderate to excellent enantioselectivity and high yields. A series of ß-nitrostyrenes reacted with pyruvate in the presence of NahE to give, after oxidative decarboxylation, ß-aryl-γ-nitrobutyric acids in up to 99 % yield without need for chromatography, providing a simple preparative-scale route to chiral GABA analogues. This reaction represents the first example of an aldolase displaying promiscuous Michaelase activity and opens the use of nitroalkenes in place of aldehydes as substrates for aldolases.

Phosphonate and α-Fluorophosphonate Analogues of d-Glucose 6-Phosphate as Active-Site Probes of 1l-myo-Inositol 1-Phosphate Synthase.

The biosynthesis of myo-inositol (mI) is central to the function of many organisms across all kingdoms of life. The first and rate-limiting step in this pathway is catalyzed by 1l-myo-inositol 1-phosphate synthase (mIPS), which converts d-glucose 6-phosphate (G6P) into 1l-myo-inositol 1-phosphate (mI1P). Extensive studies have shown that this reaction occurs through a stepwise NAD+-dependent redox aldol cyclization mechanism producing enantiomerically pure mI1P. Although the stereochemical nature of the mechanism has been elucidated, there is a lack of understanding of the importance of amino acid residues in the active site. Crystal structures of mIPS in the ternary complex with substrate analogues and NAD(H) show different ligand orientations. We therefore proposed to use isosteric and isoelectronic analogues of G6P to probe the active site. Here, we report the synthesis of the methylenephosphonate, difluoromethylenephosphonate, and (R)- and (S)-monofluoromethylenephosphonate analogues of G6P and their evaluation as inhibitors of mIPS activity. While the CH2 and CF2 analogues were produced with slight modification of a previously described route, the CHF analogues were synthesized through a new, shorter pathway. Kinetic behavior shows that all compounds are reversible competitive inhibitors with respect to G6P, with Ki values in the order CF2 (0.18 mM) < (S)-CHF (0.24 mM) < (R)-CHF (0.59 mM) < CH2 (1.2 mM). Docking studies of these phosphonates using published crystal structures show that substitution of the oxygen atom of the substrate changes the conformation of the resulting inhibitors, altering the position of carbon-6 and carbon-5, and this change is more pronounced with fluorine substitution.

Phosphoenolpyruvate Mutase-Catalyzed C-P Bond Formation: Mechanistic Ambiguities and Opportunities.

Phosphonates are produced across all domains of life and used widely in medicine and agriculture. Biosynthesis almost universally originates from the enzyme phosphoenolpyruvate mutase (Ppm), EC 5.4.2.9, which catalyzes O-P bond cleavage in phosphoenolpyruvate (PEP) and forms a high energy C-P bond in phosphonopyruvate (PnPy). Mechanistic scrutiny of this unusual intramolecular O-to-C phosphoryl transfer began with the discovery of Ppm in 1988 and concluded in 2008 with computational evidence supporting a concerted phosphoryl transfer via a dissociative metaphosphate-like transition state. This mechanism deviates from the standard 'in-line attack' paradigm for enzymatic phosphoryl transfer that typically involves a phosphoryl-enzyme intermediate, but definitive evidence is sparse. Here we review the experimental evidence leading to our current mechanistic understanding and highlight the roles of previously underappreciated conserved active site residues. We then identify remaining opportunities to evaluate overlooked residues and unexamined substrates/inhibitors.

Characterization of an α-Glucosidase Enzyme Conserved in Gardnerella spp. Isolated from the Human Vaginal Microbiome.

Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, in which a lactobacillus-dominated community is replaced with mixed bacteria, including Gardnerella species. Co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, including glycogen, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 genomes demonstrated that a putative α-glucosidase-encoding sequence, CG400_06090, was conserved in all Gardnerella spp. The predicted α-glucosidase enzyme was expressed, purified, and functionally characterized. The enzyme was active on a variety of maltooligosaccharides with maximum activity at pH 7. Km, kcat, and kcat/Km values for the substrate 4-nitrophenyl α-d-glucopyranoside were 8.3 µM, 0.96 min-1, and 0.11 µM-1 min-1, respectively. Glucose was released from maltose, maltotriose, maltotetraose, and maltopentaose, but no products were detected when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an α-glucosidase enzyme that may contribute to the multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major carbon source available to vaginal bacteria. The significance of our research is characterizing the activity of an enzyme conserved in Gardnerella species that likely contributes to the ability of these bacteria to utilize glycogen.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue.

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Snapshots along the catalytic path of KabA, a PLP-dependent aminotransferase required for kanosamine biosynthesis in Bacillus cereus UW85.

Kanosamine is an antibiotic and antifungal monosaccharide. The kanosamine biosynthetic pathway from glucose 6-phosphate in Bacillus cereus UW85 was recently reported, and the functions of each of the three enzymes in the pathway, KabA, KabB and KabC, were demonstrated. KabA, a member of a subclass of the VIß family of PLP-dependent aminotransferases, catalyzes the second step in the pathway, generating kanosamine 6-phosphate (K6P) using l-glutamate as the amino-donor. KabA catalysis was shown to be extremely efficient, with a second-order rate constant with respect to K6P transamination of over 107 M-1s-1. Here we report the high-resolution structure of KabA in both the PLP- and PMP-bound forms. In addition, co-crystallization with K6P allowed the structure of KabA in complex with the covalent PLP-K6P adduct to be solved. Co-crystallization or soaking with glutamate or 2-oxoglutarate did not result in crystals with either substrate/product. Reduction of the PLP-KabA complex with sodium cyanoborohydride gave an inactivated enzyme, and crystals of the reduced KabA were soaked with the l-glutamate analog glutarate to mimic the KabA-PLP-l-glutamate complex. Together these four structures give a complete picture of how the active site of KabA recognizes substrates for each half-reaction. The KabA structure is discussed in the context of homologous aminotransferases.

Preparation and Application of 13C-Labeled myo-Inositol to Identify New Catabolic Products in Inositol Metabolism in Lactobacillus casei.

myo-Inositol (mI) is widely distributed in all domains of life and is important for several cellular functions, including bacterial survival. The enzymes responsible for the bacterial catabolism of mI, encoded in the iol operon, can vary from one organism to another, and these pathways have yet to be fully characterized. We previously identified a new scyllo-inositol dehydrogenase (sIDH) in the iol operon of Lactobacillus casei that can oxidize mI in addition to the natural substrate, scyllo-inositol, but the product of mI oxidation was not determined. Here we report the identification of these metabolites by monitoring the reaction with 13C nuclear magnetic resonance. We prepared all six singly 13C-labeled mI isotopomers through a biocatalytic approach and used these labeled inositols as substrates for sIDH. The use of all six singly labeled mI isotopomers allowed for metabolite characterization without isolation steps. sIDH oxidation of mI produces 1l-5-myo-inosose preferentially, but also two minor products, 1d-chiro-inosose and 1l-chiro-inosose. Together with previous crystal structure data for sIDH, we were able to rationalize the observed oxidation preference. Our relatively simple procedure for the preparation of isotopically labeled mI standards can have broad applications for the study of mI biotransformations.

Asparagine-84, a regulatory allosteric site residue, helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.

The kanosamine biosynthetic pathway in Bacillus cereus UW85: Functional and kinetic characterization of KabA, KabB, and KabC.

Kanosamine is an aminosugar antibiotic, and component of complex antibiotics such as kanamycin. The biosynthesis of kanosamine varies among different bacteria; best known is a pathway starting from UDP-glucose, but Bacillus subtilis can produce kanosamine in a three-step pathway from glucose 6-phosphate. A set of genes proposed to encode a kanosamine pathway has previously been identified within the zwittermicin A gene cluster of Bacillus cereus UW85. These genes, designated kabABC, are similar to the B. subtilis kanosamine pathway genes (ntdABC), but have never been studied experimentally. We have expressed each of the kab genes, and studied the in vitro substrate scope and reaction rates and kinetic mechanisms of all three enzymes. The kab genes encode enzymes that catalyze a route similar to that found in B. subtilis from glucose 6-phosphate to kanosamine, passing through an unusual and unstable 3-keto intermediate. Kinetic studies show the first step in the pathway, the KabC-catalyzed oxidation of glucose 6-phosphate at carbon-3, is very slow relative to the subsequent KabA-catalyzed aminotransferase and KabB-catalyzed phosphatase reactions. KabC differs from its homolog, NtdC, in that it is NADP- rather than NAD-dependent. The KabA kinetic study is the first such report for a kanosamine 6-phosphate aminotransferase, revealing an extremely efficient PLP-dependent reaction. These results show that this kanosamine biosynthesis pathway occurs in more than one organism, and that the reactions are tuned in order to avoid any accumulation of the unstable intermediate.

Simultaneous Measurement of Glucose-6-phosphate 3-Dehydrogenase (NtdC) Catalysis and the Nonenzymatic Reaction of Its Product: Kinetics and Isotope Effects on the First Step in Kanosamine Biosynthesis.

Glucose-6-phosphate 3-dehydrogenase (NtdC) is an NAD-dependent oxidoreductase encoded in the NTD operon of Bacillus subtilis. The oxidation of glucose 6-phosphate by NtdC is the first step in kanosamine biosynthesis. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated. The NtdC-catalyzed reaction is very slow at low and neutral pH, and its rate increases to a maximum near pH 9.5. However, under alkaline conditions, the product is not stable because of ring opening followed by deprotonation of the 1,3-dicarbonyl compound. The absorbance band due to this enolate at 310 nm overlaps with that of the other enzymatic product, NADH, complicating kinetic measurements. We report the deconvolution of the resulting spectra of the reaction to determine the rate constants and likely kinetic mechanism. In doing so, we were able to determine the extinction coefficient of the enolate of 3oG6P (23000 M-1 cm-1), which allowed the measurement of the first-order rate constant (5.51 × 10-3 s-1) and activation energy (93 kJ mol-1) of nonenzymatic enolate formation. Using deuterium-labeled substrates, we show that hydride transfer from carbon 3 is partially rate-limiting in the enzymatic reaction, and deuterium substitution on carbon 2 has no significant effect on the enzymatic reaction but lowers the rate of deprotonation of 3oG6P 4-fold. These experiments clearly establish the regiochemistry of the reactions. Coupling of the NtdC reaction with the subsequent step in the pathway, NtdA-catalyzed glutamate-dependent amino transfer, has a small but significant effect on the rate of NAD reduction, consistent with these enzymes working together to process the unstable metabolite.

Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase.

Dihydrodipicolinate synthase is a tetrameric enzyme of the diaminopimelate pathway in bacteria and plants. The protein catalyzes the condensation of pyruvate (Pyr) and aspartate semialdehyde en route to the end product lysine (Lys). Dihydrodipicolinate synthase from Campylobacter jejuni (CjDHDPS) is allosterically inhibited by Lys. CjDHDPS is a promising antibiotic target, as highlighted by the recent development of a potent bis-lysine (bisLys) inhibitor. The mechanism whereby Lys and bisLys allosterically inhibit CjDHDPS remains poorly understood. In contrast to the case for other allosteric enzymes, crystallographically detectable conformational changes in CjDHDPS upon inhibitor binding are very minor. Also, it is difficult to envision how Pyr can access the active site; the available X-ray data seemingly imply that each turnover step requires diffusion-based mass transfer through a narrow access channel. This study employs hydrogen/deuterium exchange mass spectrometry for probing the structure and dynamics of CjDHDPS in a native solution environment. The deuteration kinetics reveal that the most dynamic protein regions are in the direct vicinity of the substrate access channel. This finding is consistent with the view that transient opening/closing fluctuations facilitate access of the substrate to the active site. Under saturating conditions, both Lys and bisLys cause dramatically reduced dynamics in the inhibitor binding region. In addition, rigidification extends to regions close to the substrate access channel. This finding strongly suggests that allosteric inhibitors interfere with conformational fluctuations that are required for CjDHDPS substrate turnover. In particular, our data imply that Lys and bisLys suppress opening/closing events of the access channel, thereby impeding diffusion of the substrate into the active site. Overall, this work illustrates why allosteric control does not have to be associated with crystallographically detectable large-scale transitions. Our experiments provide evidence that in CjDHDPS allostery is mediated by changes in the extent of thermally activated conformational fluctuations.

Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.

Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and ß-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development.

Tyrosine 110 plays a critical role in regulating the allosteric inhibition of Campylobacter jejuni dihydrodipicolinate synthase by lysine.

Dihydrodipicolinate synthase (DHDPS), an enzyme found in most bacteria and plants, controls a critical step in the biosynthesis of l-lysine and meso-diaminopimelate, necessary components for bacterial cell wall biosynthesis. DHDPS catalyzes the condensation of pyruvate and (S)-aspartate-ß-semialdehyde, forming an unstable product that is dehydrated to dihydrodipicolinate. The tetrameric enzyme is allosterically inhibited by l-lysine, and a better understanding of the allosteric inhibition mechanism is necessary for the design of potent antibacterial therapeutics. Here we describe the high-resolution crystal structures of DHDPS from Campylobacter jejuni with and without its inhibitor bound to the allosteric sites. These structures reveal a role for Y110 in the regulation of the allosteric inhibition by lysine. Mutation of Y110 to phenylalanine results in insensitivity to lysine inhibition, although the mutant crystal structure reveals that lysine does bind in the allosteric site. Comparison of the lysine-bound Y110F structure with wild-type structures reveals that key structural changes due to lysine binding are absent in this mutant.

The structure of NtdA, a sugar aminotransferase involved in the kanosamine biosynthetic pathway in Bacillus subtilis, reveals a new subclass of aminotransferases.

NtdA from Bacillus subtilis is a sugar aminotransferase that catalyzes the pyridoxal phosphate-dependent equatorial transamination of 3-oxo-α-D-glucose 6-phosphate to form α-D-kanosamine 6-phosphate. The crystal structure of NtdA shows that NtdA shares the common aspartate aminotransferase fold (Type 1) with residues from both monomers forming the active site. The crystal structures of NtdA alone, co-crystallized with the product α-D-kanosamine 6-phosphate, and incubated with the amine donor glutamate reveal three key structures in the mechanistic pathway of NtdA. The structure of NtdA alone reveals the internal aldimine form of NtdA with the cofactor pyridoxal phosphate covalently attached to Lys-247. The addition of glutamate results in formation of pyridoxamine phosphate. Co-crystallization with kanosamine 6-phosphate results in the formation of the external aldimine. Only α-D-kanosamine 6-phosphate is observed in the active site of NtdA, not the ß-anomer. A comparison of the structure and sequence of NtdA with other sugar aminotransferases enables us to propose that the VIß family of aminotransferases should be divided into subfamilies based on the catalytic lysine motif.

Dihydrodipicolinate synthase from Campylobacter jejuni: kinetic mechanism of cooperative allosteric inhibition and inhibitor-induced substrate cooperativity.

Dihydrodipicolinate synthase (DHDPS), an enzyme of the meso-diaminopimelate pathway of lysine biosynthesis, is essential for bacterial growth and is considered a target for novel antibiotics. We have studied DHDPS from Campylobacter jejuni for the first time, determining the kinetic mechanism of catalysis and inhibition with its natural allosteric feedback inhibitor (S)-lysine. The tetrameric enzyme is known to have two allosteric sites, each of which binds two molecules of lysine. The results suggest that lysine binds highly cooperatively, and primarily to the F form of the enzyme during the ping-pong mechanism. By applying graphical methods and nonlinear regression, we have discriminated between the possible kinetic models and determined the kinetic and inhibition constants and Hill coefficients. We conclude that (S)-lysine is an uncompetitive partial inhibitor with respect to its first substrate, pyruvate, and a mixed partial inhibitor with respect to its second substrate, (S)-aspartate-ß-semialdehyde (ASA), which differs from the kinetic models for inhibition reported for DHDPS from other sources. The Hill coefficients for the binding of lysine to different forms of the enzyme are all greater than 2, suggesting that the two allosteric sites are not independent. It has been found that ASA binds cooperatively in the presence of (S)-lysine, and the cooperativity of binding increases at near-KM concentrations of pyruvate. The incorporation of Hill coefficients into the kinetic equations was crucial for determining the kinetic model for this enzyme.

Converting NAD-specific inositol dehydrogenase to an efficient NADP-selective catalyst, with a surprising twist.

myo-Inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis converts myo-inositol to scyllo-inosose and is strictly dependent on NAD for activity. We sought to alter the coenzyme specificity to generate an NADP-dependent enzyme in order to enhance our understanding of coenzyme selectivity and to create an enzyme capable of recycling NADP in biocatalytic processes. Examination of available structural information related to the GFO/MocA/IDH family of dehydrogenases and precedents for altering coenzyme selectivity allowed us to select residues for substitution, and nine single, double, and triple mutants were constructed. Mutagenesis experiments with B. subtilis IDH proved extremely successful; the double mutant D35S/V36R preferred NADP to NAD by a factor of 5. This mutant is an excellent catalyst with a second-order rate constant with respect to NADP of 370 000 s⁻¹ M⁻¹, and the triple mutant A12K/D35S/V36R had a value of 570 000 s⁻¹ M⁻¹, higher than that of the wild-type IDH with NAD. The high-resolution X-ray crystal structure of the double mutant A12K/D35S was solved in complex with NADP. Surprisingly, the binding of the coenzyme is altered such that although the nicotinamide ring maintains the required position for catalysis, the coenzyme has twisted by nearly 90°, so the adenine moiety no longer binds to a hydrophobic cleft in the Rossmann fold as in the wild-type enzyme. This change in binding conformation has not previously been observed in mutated dehydrogenases.

A previously unrecognized kanosamine biosynthesis pathway in Bacillus subtilis.

The ntd operon in Bacillus subtilis is essential for biosynthesis of 3,3'-neotrehalosadiamine (NTD), an unusual nonreducing disaccharide reported to have antibiotic properties. It has been proposed that the three enzymes encoded within this operon, NtdA, NtdB, and NtdC, constitute a complete set of enzymes required for NTD synthesis, although their functions have never been demonstrated in vitro. We now report that these enzymes catalyze the biosynthesis of kanosamine from glucose-6-phosphate: NtdC is a glucose-6-phosphate 3-dehydrogenase, NtdA is a pyridoxal phosphate-dependent 3-oxo-glucose-6-phosphate:glutamate aminotransferase, and NtdB is a kanosamine-6-phosphate phosphatase. None of these enzymatic reactions have been reported before. This pathway represents an alternate route to the previously reported pathway from Amycolatopsis mediterranei which derives kanosamine from UDP-glucose.

Homoisocitrate dehydrogenase from Candida albicans: properties, inhibition, and targeting by an antifungal pro-drug.

The LYS12 gene from Candida albicans, coding for homoisocitrate dehydrogenase was cloned and expressed as a His-tagged protein in Escherichia coli. The purified gene product catalyzes the Mg(2+)- and K(+)-dependent oxidative decarboxylation of homoisocitrate to α-ketoadipate. The recombinant enzyme demonstrates strict specificity for homoisocitrate. SDS-PAGE of CaHIcDH revealed its molecular mass of 42.6 ± 1 kDa, whereas in size-exclusion chromatography, the enzyme eluted in a single peak corresponding to a molecular mass of 158 ± 3 kDa. Native electrophoresis showed that CaHIcDH may exist as a monomer and as a tetramer and the latter form is favored by homoisocitrate binding. CaHIcDH is an hysteretic enzyme. The K(M) values of the purified His-tagged enzyme for NAD(+) and homoisocitrate were 1.09 mM and 73.7 µM, respectively, and k(cat) was 0.38 s(-1). Kinetic parameters determined for the wild-type CaHIcDH were very similar. The enzyme activity was inhibited by (2R,3S)-3-(p-carboxybenzyl)malate (CBMA), with IC(50) = 3.78 mM. CBMA demonstrated some moderate antifungal activity in minimal media that could be enhanced upon conversion of the enzyme inhibitor into its trimethyl ester derivative (TMCBMA). TMCBMA is the first reported antifungal for which an enzyme of the AAP was identified as a molecular target.

Phosphonate and α-fluorophosphonate analogs of d-glucose 6-phosphate as active-site probes of 1l-myo-inositol 1-phosphate synthase.

Phosphate ester analogs in which the bridging oxygen is replaced with a methylene or fluoromethylene group are well known non-hydrolyzable mimics of use as inhibitors and substrate analogs for reactions involving phosphate esters. Properties of the replaced oxygen are often best mimicked by a mono-fluoromethylene group, but such groups are challenging to synthesize and can exist as two stereoisomers. Here, we describe the protocol for our method of synthesizing the α-fluoromethylene analogs of d-glucose 6-phosphate (G6P), as well as the methylene and difluoromethylene analogs, and their application in the study of 1l-myo-inositol-1-phosphate synthase (mIPS). mIPS catalyzes the synthesis of 1l-myo-inositol 1-phosphate (mI1P) from G6P, in an NAD-dependent aldol cyclization. Its key role in myo-inositol metabolism makes it a putative target for the treatment of several health disorders. The design of these inhibitors allowed for the possibility of substrate-like behavior, reversible inhibition, or mechanism-based inactivation. In this chapter, the synthesis of these compounds, expression and purification of recombinant hexahistidine-tagged mIPS, the mIPS kinetic assay and methods for determining the behavior of the phosphate analogs in the presence of mIPS, and a docking approach to rationalizing the observed behavior are described.

Using substrate analogues to probe the kinetic mechanism and active site of Escherichia coli MenD.

MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.