Structural insights of the MenD from Escherichia coli reveal ThDP affinity

MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate) synthase belongs to the superfamily of thiamin diphosphate-dependent decarboxylases, which converts isochorismate and 2-oxoglutarate to SHCHC, pyruvate, and carbon dioxide. Here, we report the first crystal structure of apo-MenD from Escherichia coli determined in tetragonal crystal form. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes. Analytical gel filtration shows that EcMenD behaves as a dimer as well as a tetramer. Circular dichroism and isothermal calorimetry results confirm EcMenD dependency on ThDP, which concomitantly helps to stabilize with better configuration.     

Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity

Menaquinone is an electron carrier in the respiratory chain of Escherichia coli during anaerobic growth. Its biosynthesis involves (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) as an intermediate, which is believed to be derived from isochorismate and 2-ketoglutarate by one of the biosynthetic enzymes-MenD. However, we found that the genuine MenD product is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC), rather than SHCHC. This is supported by the following findings: (i) isochorismate consumption and SHCHC formation are not synchronized in the enzymic reaction, (ii) the rate of SHCHC formation is independent of the enzyme concentration, (iii) SHCHC is not formed in weakly acidic or neutral solutions in which the isochorismate substrate is readily consumed by MenD, and (iv) the MenD turnover product, formed under conditions disabling SHCHC formation, possesses spectroscopic characteristics consistent with the structure of SEPHCHC and spontaneously undergoes 2,5-elimination to form SHCHC and pyruvate in weakly basic solutions. Two properties of the intermediate, ultraviolet transparency and chemical instability, provide a rationale for the fact that SHCHC has been consistently mistaken as the MenD product. In accordance with these findings, MenD was rediscovered to be a highly efficient enzyme with a high second-order rate constant and should be renamed SEPHCHC synthase. Intriguingly, the enzymatic activity responsible for conversion of SEPHCHC into SHCHC appears not to associate with any of the known enzymes in menaquinone biosynthesis but is present in the crude extract of E. coli K12, suggesting that a genuine SHCHC synthase remains to be identified to fully elucidate the ubiquitous biosynthetic pathway.     

Exploiting the high-resolution crystal structure of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> MenH to gain insight into enzyme activity

Background  

MenH (2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase) is a key enzyme in the biosynthesis of menaquinone, catalyzing an unusual 2,5-elimination of pyruvate from 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate.     

Steady-state kinetics and molecular evolution of Escherichia coli MenD [(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase], an anomalous thiamin diphosphate-dependent decarboxylase-carboligase

(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide. The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others. However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate. Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction. The protein is a dimer and shows cooperativity with respect to both substrates. The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD. MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp. NRC-1 and the eukaryote Arabidopsis thaliana. Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP. A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis. There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.     

MenD as a versatile catalyst for asymmetric synthesis

The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) from Escherichia coli K12, formerly known as SHCHC-synthase, catalyses the decarboxylation of α-ketoglutarate and the subsequent addition of the resulting succinyl-THDP to isochorismate. Here, the enzyme is tested for unphysiologial C–C bond-forming reactions.Condensation of α-ketoglutarate after decarboxylation to a broad range of aldehydes gave α-hydroxyketones with isolated yields from 26 to 87% and 94 to 98% ee for addition to aromatic aldehydes. MenD accepts a wide range of aldehydes as acceptor substrates to produce chiral α-hydroxyketones with conserved regioselectivity where the activated succinylsemialdehyde serves selectively as the donor. Regioselectivity is inverted only for condensation of α-ketoglutarate with pyruvate (activated acetaldehyde) as donor. Besides α-ketoglutarate, pyruvate and oxalacetate are accepted as donors in combination with benzaldehyde and 2-fluorobenzaldehyde as acceptors, however with decreased activity of C–C bond formation.The physiological 1,4-addition of α-ketoglutarate to isochorismate was investigated for acceptor substrate variability. (2S,3S)-2,3-Dihydroxy-2,3-dihydrobenzoate (2,3-CHD), which lacks the pyruvyl found in isochorismate, is converted to (5S,6S)-2-succinyl-5,6-dihydroxycyclohex-2-enecarboxylate. In contrast to the addition to carbonyls, the active site of MenD does appear to impose specific constraints on the acceptor substrate for 1,4-addition with α,β-unsaturated carboxylic acids.     

Menaquinone (vitamin K2) biosynthesis: evidence that the Escherichia coli menD gene encodes both 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase and alpha-ketoglutarate decarboxylase activities.

The formation of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC), the first identified intermediate in the menaquinone biosynthetic pathway, requires two reactions. They are the decarboxylation of alpha-ketoglutarate by an alpha-ketoglutarate decarboxylase, which results in the formation of succinic semialdehyde-thiamine PPi (TPP) anion, and the addition of the succinic semialdehyde-TPP anion to isochorismate carried out by the enzyme SHCHC synthase. Evidence is provided to support the conclusion that both enzymatic activities are encoded by an extended menD gene which is capable of generating a bifunctional 69-kDa protein. Consistent with the requirement for TPP in the decarboxylation of alpha-ketoglutarate, the translated amino acid sequence contains the characteristic TPP-binding motif present in all well-characterized TPP-requiring enzymes.     

Vitamin K (menaquinone) biosynthesis in bacteria: purification and probable structure of an intermediate prior to o-succinylbenzoate

The first aromatic intermediate in the menaquinone biosynthetic pathway is o-succinylbenzoate (OSB); it is formed from chorismate/isochorismate and 2-ketoglutarate. Cell-free extracts of menD+ E. coli strains synthesize an intermediate, "X", which is converted to OSB by extracts of menC+ cells. "X" has been purified to near homogeneity by HPLC. On treatment with acid, it yields both OSB and succinylbenzene (SB). This and other data, suggest that "X" has the structure, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (I).     

Biosynthesis of o-succinylbenzoic acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the alpha-ketoglutarate dehydrogenase complex.

The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity.     

Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli

Menaquinone is a lipid-soluble molecule that plays an essential role as an electron carrier in the respiratory chain of many bacteria. We have previously shown that its biosynthesis in Escherichia coli involves a new intermediate, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and requires an additional enzyme to convert this intermediate into (1 R,6 R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). Here, we report the identification and characterization of MenH (or YfbB), an enzyme previously proposed to catalyze a late step in menaquinone biosynthesis, as the SHCHC synthase. The synthase catalyzes a proton abstraction reaction that results in 2,5-elimination of pyruvate from SEPHCHC and the formation of SHCHC. It is an efficient enzyme ( k cat/ K M = 2.0 x 10 (7) M (-1) s (-1)) that provides a smaller transition-state stabilization than other enzymes catalyzing proton abstraction from carbon acids. Despite its lack of the proposed thioesterase activity, the SHCHC synthase is homologous to the well-characterized C-C bond hydrolase MhpC. The crystallographic structure of the Vibrio cholerae MenH protein closely resembles that of MhpC and contains a Ser-His-Asp triad typical of serine proteases. Interestingly, this triad is conserved in all MenH proteins and is essential for the SHCHC synthase activity. Mutational analysis found that the catalytic efficiency of the E. coli protein is reduced by 1.4 x 10 (3), 2.1 x 10 (5), and 9.3 x 10 (3) folds when alanine replaces serine, histidine, and aspartate of the triad, respectively. These results show that the SHCHC synthase is closely related to alpha/beta hydrolases but catalyzes a reaction mechanistically distinct from all known hydrolase reactions.     

Loss of phylloquinone in Chlamydomonas affects plastoquinone pool size and photosystem II synthesis

Phylloquinone functions as the electron transfer cofactor at the A(1) site of photosystem I. We have isolated and characterized a mutant of Chlamydomonas reinhardtii, menD1, that is deficient in MenD, which encodes 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, an enzyme that catalyzes the first specific step of the phylloquinone biosynthetic pathway. The mutant is photosynthetically active but light-sensitive. Analysis of total pigments by mass spectrometry reveals that phylloquinone is absent in menD1, but plastoquinone levels are not affected. This is further confirmed by the rescue of menD1 by addition of phylloquinone to the growth medium. Analysis of electron transfer by absorption spectroscopy indicates that plastoquinone replaces phylloquinone in photosystem I and that electron transfer from A(1) to the iron-sulfur centers is slowed down at least 40-fold. Consistent with a replacement of phylloquinone by plastoquinone, the size of the free plastoquinone pool of menD1 is reduced by 20-30%. In contrast to cyanobacterial MenD-deficient mutants, photosystem I accumulates normally in menD1, whereas the level of photosystem II declines. This decrease is because of reduced synthesis of the photosystem II core subunits. The relationship between plastoquinone occupancy of the A(1) site in photosystem I and the reduced accumulation of photosystem II is discussed.     

Sequence and overexpression of the menD gene from Escherichia coli.

The menD gene of Escherichia coli codes for the first enzyme of menaquinone biosynthesis, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase. DNA sequence analysis of menD shows an open reading frame encoding a 52-kilodalton protein. Possible promoter and ribosome binding sites are present. Insertion of the menD gene into a tac promoter expression vector leads to nearly a 100-fold increase in the level of SHCHC synthase activity upon induction with isopropyl-beta-D-thiogalactoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins shows a 61-kilodalton protein produced upon induction of the menD-containing expression vector. This is the first reported sequence analysis of a men gene and the first significant amplification of any of the menaquinone biosynthetic enzymes.     

Vitamin K (menaquinone) biosynthesis in bacteria: high-performance liquid chromatographic assay of the overall synthesis of o-succinylbenzoic acid and of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase

An early enzyme in menaquinone (vitamin K2) biosynthesis is the synthase forming 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) from isochorismic acid. In turn, SHCHC is aromatized to o-succinylbenzoic acid (OSB) by OSB synthase. An assay for the combined activity of these two enzymes ("overall OSB synthesis") has been developed using a high-performance liquid chromatographic method for the quantitation of OSB. The assay, which measures as little as 0.1 nmol of OSB, is vastly superior to the radiogas chromatographic method previously used to estimate overall OSB synthesis. To measure SHCHC synthase activity separately, the enzymatically formed SHCHC is converted nonenzymatically to OSB (heating to 80 degrees C, pH 10, 10 min), which is then quantitated by the HPLC assay. The preparation of the substrate, isochorismic acid, and its purification by preparative HPLC are also described.     

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.     

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.     

A protecting group-free synthesis of deazathiamine: a step toward inhibitor design

The discovery of 3-deazathiamine diphosphate (deazaThDP) as a potent inhibitor analog of the cofactor thiamine diphosphate (ThDP) has highlighted the need for an efficient and scalable synthesis of deazaThDP. Such a method would facilitate development of analogs with the ability to inhibit individual ThDP-dependent enzymes selectively. Toward the goal of developing selective inhibitors of the mycobacterial enzyme 2-hydroxy-3-oxoadipate synthase (HOAS), we report an improved synthesis of deazaThDP without use of protecting groups. Tribromo-3-methylthiophene served as a versatile starting material whose selective functionalization permitted access to deazaThDP in five steps, with potential to make other analogs accessible in substantial amounts.     

The menD and menE homologs code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate synthase and O-succinylbenzoic acid-CoA synthase in the phylloquinone biosynthetic pathway of Synechocystis sp. PCC 6803

The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains genes identified as menD and menE, homologs of Escherichia coli genes that code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase and O-succinylbenzoic acid-CoA ligase in the menaquinone biosynthetic pathway. In cyanobacteria, the product of this pathway is 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone), a molecule used exclusively as an electron transfer cofactor in Photosystem (PS) I. The menD(-) and menE(-) strains were generated, and both were found to lack phylloquinone. Hence, no alternative pathways exist in cyanobacteria to produce O-succinylbenzoyl-CoA. Q-band EPR studies of photoaccumulated quinone anion radical and optical kinetic studies of the P700(+) [F(A)/F(B)](-) backreaction indicate that in the mutant strains, plastoquinone-9 functions as the electron transfer cofactor in the A(1) site of PS I. At a light intensity of 40 microE m(-2) s(-1), the menD(-) and menE(-) mutant strains grew photoautotrophically and photoheterotrophically, but with doubling times slower than the wild type. Both of which are sensitive to high light intensities. Low-temperature fluorescence studies show that in the menD(-) and menE(-) mutants, the ratio of PS I to PS II is reduced relative to the wild type. Whole-chain electron transfer rates in the menD(-) and menE(-) mutant cells are correspondingly higher on a chlorophyll basis. The slower growth rate and high-light sensitivity of the menD(-) and menE(-) mutants are therefore attributed to a lower content of PS I per cell.     

Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli

o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2'-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.     

Crystal Structures of E. coli Native MenH and Two Active Site Mutants

Recent revision of the biosynthetic pathway for menaquinone has led to the discovery of a previously unrecognized enzyme 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, also known as MenH. This enzyme has an α/β hydrolase fold with a catalytic triad comprising Ser86, His232, and Asp210. Mutational studies identified a number of conserved residues of importance to activity, and modeling further implicated the side chains of Tyr85 and Trp147 in formation of a non-standard oxyanion hole. We have solved the structure of E. coli MenH (EcMenH) at 2.75 Å resolution, together with the structures of the active site mutant proteins Tyr85Phe and Arg124Ala, both at 2.5 Å resolution. EcMenH has the predicted α/β hydrolase fold with its core α/β domain capped by a helical lid. The active site, a long groove beneath the cap, contains a number of conserved basic residues and is found to bind exogeneous anions, modeled as sulfate and chloride, in all three crystal structures. Docking studies with the MenH substrate and a transition state model indicate that the bound anions mark the binding sites for anionic groups on the substrate. The docking studies, and careful consideration of the active site geometry, further suggest that the oxyanion hole is of a conventional nature, involving peptide NH groups, rather than the proposed site involving Tyr85 and Trp147. This is in accord with conclusions from the structure of S. aureus MenH. Comparisons with the latter do, however, indicate differences in the periphery of the active site that could be of relevance to selective inhibition of MenH enzymes.     

Structural and functional analysis of Vitamin K2 synthesis protein MenD

Here we describe in detail the crystal structures of the Vitamin K₂ synthesis protein MenD, from Escherichia coli, in complex with thiamine diphosphate (ThDP) and oxoglutarate, and the effects of cofactor and substrate on its structural stability. This is the first reported structure of MenD in complex with oxoglutarate. The residues Gly472 to Phe488 of the active site region are either disordered, or in an open conformation in the MenD oxoglutarate complex structure, but adopt a closed conformation in the MenD ThDP complex structure. Biospecific-interaction analysis using surface plasmon resonance (SPR) technology reveals an affinity for ThDP and oxoglutarate in the nanomolar range. Biochemical and structural analysis confirmed that MenD is highly dependent on ThDP for its structural stability. Our structural results combined with the biochemical assay reveal novel features of the enzyme that could be utilized in a program of rational structure-based drug design, as well as in helping to enhance our knowledge of the menaquinone synthesis pathway in greater detail.