David R. J. Palmer

Methanogen Homoaconitase Catalyzes Both Hydrolyase Reactions in Coenzyme B Biosynthesis

Homoaconitase enzymes catalyze hydrolyase reactions in the α-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis. Despite the homology of this iron-sulfur protein to aconitase, previously studied homoaconitases catalyze only the hydration of cis-homoaconitate to form homoisocitrate rather than the complete isomerization of homocitrate to homoisocitrate. The MJ1003 and MJ1271 proteins from the methanogen Methanocaldococcus jannaschii formed the first homoaconitase shown to catalyze both the dehydration of (R)-homocitrate to form cis-homoaconitate, and its hydration is shown to produce homoisocitrate. This heterotetrameric enzyme also used the analogous longer chain substrates cis-(homo)2aconitate, cis-(homo)3aconitate, and cis-(homo)4aconitate, all with similar specificities. A combination of the homoaconitase with the M. jannaschii homoisocitrate dehydrogenase catalyzed all of the isomerization and oxidative decarboxylation reactions required to form 2-oxoadipate, 2-oxopimelate, and 2-oxosuberate, completing three iterations of the 2-oxoacid elongation pathway. Methanogenic archaeal homoaconitases and fungal homoaconitases evolved in parallel in the aconitase superfamily. The archaeal homoaconitases share a common ancestor with isopropylmalate isomerases, and both enzymes catalyzed the hydration of the minimal substrate maleate to form d-malate. The variation in substrate specificity among these enzymes correlated with the amino acid sequences of a flexible loop in the small subunits.

Succinylphosphonate Esters Are Competitive Inhibitors of MenD That Show Active-Site Discrimination between Homologous α-Ketoglutarate-Decarboxylating Enzymes

MenD is a thiamin diphosphate-dependent enzyme catalyzing the first unique step in menaquinone biosynthesis in bacteria. We have synthesized acylphosphonate ester analogues of alpha-ketoglutarate, a substrate of MenD. These compounds are competitive inhibitors of MenD, with K(i) values as low as 700 nM. Observed structure-activity relationships are in notable contrast to those reported previously for succinylphosphonate inhibition of the alpha-ketoglutarate dehydrogenase complex, despite the apparent homology of these enzymes, and the identical decarboxylation reactions catalyzed. Inhibiting menaquinone biosynthesis is a proposed approach to inhibiting Mycobacterium tuberculosis growth. These inhibitors showed no significant inhibition of M. tuberculosis growth in vitro under aerobic and hypoxic conditions but give new information about the binding characteristics of the MenD active site. Site-directed mutation of Ser391 to alanine had only a minor effect on catalysis, but even the conservative mutation of Arg395 to lysine had a significant effect on the catalytic processing of isochorismate.

Improved asymmetric syntheses of (R)-(−)-homocitrate and (2R,3S)-(−)-homoisocitrate, intermediates in the α-aminoadipate pathway of fungi

Abstract Improved asymmetric syntheses of the title compounds are reported. Both products are produced through diastereoselective alkylation of malic acid; ( R )-homocitrate synthesis uses the self-regeneration of stereocenters approach. Both procedures represent an improvement in yield over existing methods without loss of stereoselectivity.

Isothermal Titration Microcalorimetry Reveals the Cooperative and Noncompetitive Nature of Inhibition of Sinorhizobium meliloti L5-30 Dihydrodipicolinate Synthase by (S)-Lysine†

MosA, a dihydrodipicolinate synthase (DHDPS) from Sinorhizobium meliloti L5-30, catalyzes a class I aldolase reaction that is allosterically inhibited by (S)-lysine. The thermodynamics of (S)-lysine binding to apoenzyme, and to enzyme saturated with pyruvate or with 2-oxobutyrate, are evaluated here using isothermal titration microcalorimetry. Results unambiguously support a noncompetitive mechanism, with substrate-dependent differences in the energetics of inhibitor binding. Inhibition is strikingly cooperative: a second molecule of (S)-lysine binds 10(5) times more tightly than the first.

Structural, Functional and Calorimetric Investigation of MosA, a Dihydrodipicolinate Synthase from Sinorhizobium meliloti L5–30, does not Support Involvement in Rhizopine Biosynthesis

MosA is an enzyme from Sinorhizobium meliloti L5–30, a beneficial soil bacterium that forms a symbiotic relationship with leguminous plants. MosA was proposed to catalyze the conversion of scyllo‐inosamine to 3‐O‐methyl‐scyllo‐inosamine (compounds known as rhizopines), despite the MosA sequence showing a strong resemblance to dihydrodipicolinate synthase (DHDPS) sequences rather than to methyltransferases. Our laboratory has already shown that MosA is an efficient catalyst of the DHDPS reaction. Here we report the structure of MosA, solved to 1.95 Å resolution, which resembles previously reported DHDPS structures. In this structure Lys161 forms a Schiff base adduct with pyruvate, consistent with the DHDPS mechanism. We have synthesized both known rhizopines and investigated their ability to interact with MosA in the presence and absence of methyl donors. No MosA‐catalyzed methyltransferase activity is observed in the presence of scyllo‐inosamine and S‐adenosylmethionine (SAM). 2‐Oxobutyrate can form a Schiff base with MosA, acting as a competitive inhibitor of MosA‐catalyzed dihydrodipicolinate synthesis. It can be trapped on the enzyme by reaction with sodium borohydride, but does not act as a methyl donor. The presence of rhizopines does not affect the kinetics of dihydrodipicolinate synthesis. Isothermal titration calorimetry (ITC) shows no apparent interaction of MosA with rhizopines and SAM. Similar experiments with pyruvate as titrant demonstrate that the reversible Schiff base formation is largely entropically driven. This is the first use of ITC to study Schiff base formation between an enzyme and its substrate.

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.

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-β-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.

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.

Structural investigation of myo-inositol dehydrogenase from Bacillus subtilis: implications for catalytic mechanism and inositol dehydrogenase subfamily classification.

Inositol dehydrogenase from Bacillus subtilis (BsIDH) is a NAD+-dependent enzyme that catalyses the oxidation of the axial hydroxy group of myo-inositol to form scyllo-inosose. We have determined the crystal structures of wild-type BsIDH and of the inactive K97V mutant in apo-, holo- and ternary complexes with inositol and inosose. BsIDH is a tetramer, with a novel arrangement consisting of two long continuous β-sheets, formed from all four monomers, in which the two central strands are crossed over to form the core of the tetramer. Each subunit in the tetramer consists of two domains: an N-terminal Rossmann fold domain containing the cofactor-binding site, and a C-terminal domain containing the inositol-binding site. Structural analysis allowed us to determine residues important in cofactor and substrate binding. Lys97, Asp172 and His176 are the catalytic triad involved in the catalytic mechanism of BsIDH, similar to what has been proposed for related enzymes and short-chain dehydrogenases. Furthermore, a conformational change in the nicotinamide ring was observed in some ternary complexes, suggesting hydride transfer to the si-face of NAD+. Finally, comparison of the structure and sequence of BsIDH with other putative inositol dehydrogenases allowed us to differentiate these enzymes into four subfamilies based on six consensus sequence motifs defining the cofactor- and substrate-binding sites.

The Structure of NtdA, a Sugar Aminotransferase Involved in the Kanosamine Biosynthetic Pathway in Bacillus subtilis, Reveals a New Subclass of Aminotransferases.

Background: NtdA represents a novel aminotransferase recognizing a sugar 6-phosphate. Results: We have determined the structure of NtdA with pyridoxamine phosphate (the internal and external aldimines), identifying determinants of substrate specificity. Conclusion: The structures suggest a canonical aminotransferase chemical mechanism, but features exclude the binding of sugar nucleotides. Significance: A new subfamily of sugar aminotransferases is revealed, enhancing our understanding of antibiotic biosynthesis. 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 β-anomer. A comparison of the structure and sequence of NtdA with other sugar aminotransferases enables us to propose that the VIβ family of aminotransferases should be divided into subfamilies based on the catalytic lysine motif.