Glutamate Mutase Research Articles

The Co-N bond cleavage in the adenosyncobalamin cofactor in advance to glutamate mutase and methylmalonyl-CoA mutase processes

The in vivo experiments show that the adenosylcobalamin cofactor in glutamate mutase and methylmalonyl-CoA mutase processes lose its dimethylbenzimidazole axial ligand before starting the enzymatic processes. Complete active space self-consistent field geometry optimization of the vitamin B12 active forms plus substrates joint models have been performed. These joint models include the adenosylcobalamin cofactor, the carboxyl negative ion model of the studied processes’ active substrates, and the histidine molecule. Partial electronic density is transferred from the highest occupied substrate molecular orbitals to the lowest unoccupied antibonding molecular orbitals, which consist of corrin ring and dimethylbenzimidazole ligand common molecular orbitals during the multi-configurational self-consistent field molecular orbital mixing process. As a result, the Co-N axial bond is permanently elongated during the complete active space self-consistent field geometry optimization until its complete rupture and until the removal of the dimethylbenzimidazole ligand from the central cobalt atom and the corrin ring is complete. The Co-N bond cleavage in the adenosylcobalamin cofactors in the studied processes is running as no energy barrier process under the influence of their active substrates and histidine molecule.

Synthesis and structural characterization of new Schiff base probe: A conformation modulator for Co-C bond cleavage in coenzyme B12-dependent enzymes

Coenzyme B12-dependent enzymes catalyze complex radical-mediated reactions initiated by homolytic cleavage of the organometallic Co-C bond of the cofactors upon substrate binding. Herein, we synthesize new 2,3-diaminomaleonitrile (DAMN) based Schiff base probe for an investigation of binding interaction with coenzyme B12-dependent enzymes by molecular docking approach. The formulated compound has been well characterized by elemental analysis, NMR (1H and 13C), Mass spectrometry and UV-visible spectroscopy. Moreover, the complete structure elucidation of Schiff base probe was achieved via X-ray crystallography. Docking study reveals that the newly synthesized Schiff base compound confers good binding response towards glutamate mutase and methionine synthase as supported by calculated docking score and binding energies -7.7 kcal/mol and -8.9 kcal/mol, respectively. The results indicate significant binding effect of synthesized Schiff base on the catalysis of B12-dependent enzymes. We hope that the present information can be utilized to develop potent therapeutic drugs.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012-fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5′‐deoxy‐5′adenosyl radical, which originates from reversible Co−C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012‐fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate‐loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co−C bond cleavage. Strategically interacting adjacent adenosine‐ and substrate‐binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including “negative catalysis”, a paradigm for AdoCbl‐dependent mutases.

Glutamate mutase and 2-methyleneglutarate mutase.

Glutamate mutase converts ( S )-glutamate to ( S , S )-3-methylaspartate, whereas 2-methyleneglutarate mutase isomerizes 2-methyleneglutarate to ( R )-3-methylitaconate. Both enzymes occur in amino acid fermenting clostridia, require coenzyme B 12 (adenosylcobalamin) and catalyze a reversible, radical carbon skeleton rearrangement. The enzymes are assayed using the consecutive enzymes in their fermentation pathways: methylaspartase and methylitaconate isomerase, which produce the higher absorbing mesaconate and 2,3-dimethylmaleate, respectively. Although the quaternary structures of glutamate mutase and 2-methyleneglutarate mutase are different, the binding mode of coenzyme B 12 and their catalytic mechanism appear to be very similar. The crystal structure of glutamate mutase and EPR spectroscopy of both enzymes during catalysis revealed a distance of 6.6 Å between the C-H bond of the substrate to be cleaved and the Co-atom of cob(II)alamin. Methods are described for facile production of both enzymes in Escherichia coli and purification.

Computational investigations of B12-dependent enzymatic reactions.

Nature employs two biologically active forms of vitamin B12, adenosylcobalamin (or coenzyme B12) and methylcobalamin, as cofactors in molecular transformations both in bacteria and mammals. Computational chemistry, guided by experimental data, has been used to explore fundamental characteristics of these enzymatic reactions. In particular, the quantum mechanics/molecular mechanics (QM/MM) method has proven to be a powerful tool in elucidating important characteristics of B12-dependent enzymatic reactions. Herein, we will present a brief tutorial in conducting QM/MM calculations for B12 enzymatic reactions. We will summarize recent contributions that target the use of QM/MM calculations in both photochemical and enzymatic reactions including AdoCbl-dependent ethanolamine ammonia lyase, glutamate mutase, and photoreceptor CarH.

Using kinetic isotope effects to probe the mechanism of adenosylcobalamin-dependent enzymes.

Adenosylcobalamin- (AdoCbl) dependent enzyme reactions involved the transfer of hydrogen atoms between the 5'-carbon of the coenzyme and the substrates and products of the reaction. Tritium and deuterium kinetic isotope effect measurements are, therefore, a valuable tool to probe the mechanisms of AdoCbl-dependent enzymes, as they can provide information about the reaction pathway and the rate-determining step. Furthermore, if the intrinsic kinetic isotope effect can be isolated, information on the nature of the transition state associated with hydrogen transfer can be obtained. In this chapter I present methods for the preparation of isotopically-labeled AdoCbl and their use in rapid chemical quench experiments that allow isotope effects on specific steps in the reaction to be isolated. These techniques are illustrated with examples from my laboratory's studies on the AdoCbl dependent enzyme, glutamate mutase.

Energy Conservation in Fermentations of Anaerobic Bacteria.

Anaerobic bacteria ferment carbohydrates and amino acids to obtain energy for growth. Due to the absence of oxygen and other inorganic electron acceptors, the substrate of a fermentation has to serve as electron donor as well as acceptor, which results in low free energies as compared to that of aerobic oxidations. Until about 10 years ago, anaerobes were thought to exclusively use substrate level phosphorylation (SLP), by which only part of the available energy could be conserved. Therefore, anaerobes were regarded as unproductive and inefficient energy conservers. The discovery of electrochemical Na+ gradients generated by biotin-dependent decarboxylations or by reduction of NAD+ with ferredoxin changed this view. Reduced ferredoxin is provided by oxidative decarboxylation of 2-oxoacids and the recently discovered flavin based electron bifurcation (FBEB). In this review, the two different fermentation pathways of glutamate to ammonia, CO2, acetate, butyrate and H2 via 3-methylaspartate or via 2-hydroxyglutarate by members of the Firmicutes are discussed as prototypical examples in which all processes characteristic for fermentations occur. Though the fermentations proceed on two entirely different pathways, the maximum theoretical amount of ATP is conserved in each pathway. The occurrence of the 3-methylaspartate pathway in clostridia from soil and the 2-hydroxyglutarate pathway in the human microbiome of the large intestine is traced back to the oxygen-sensitivity of the radical enzymes. The coenzyme B12-dependent glutamate mutase in the 3-methylaspartate pathway tolerates oxygen, whereas 2-hydroxyglutaryl-CoA dehydratase is extremely oxygen-sensitive and can only survive in the gut, where the combustion of butyrate produced by the microbiome consumes the oxygen and provides a strict anaerobic environment. Examples of coenzyme B12-dependent eliminases are given, which in the gut are replaced by simpler extremely oxygen sensitive glycyl radical enzymes.

Kinetic Studies of a Coenzyme B12 Dependent Reaction Catalyzed by Glutamate Mutase from <i>Clostridium cochlearium</i>

The coenzyme B12 dependent glutamate mutase is composed of two apoenzyme proteins subunits; S and E2, which while either fused or separate assemble with coenzyme B12 to form an active holoenzyme (E2S2-B12) for catalyzing the reversible isomerization between (S)-glutamate and (2S, 3S)-3-methylas- partate. In order to assay the activity of glutamate mutase by UV spectrophotometry, this reaction is often coupled with methylaspartase which deaminates (2S, 3S)-3-methylaspartate to form mesaconate (λmax = 240 nm, ?240 = 3.8 mM?1?cm?1). The activities of different reconstitutions of glutamate mutase from separate apoenzyme components S and E in varied amounts of coenzyme B12 and adenosylpeptide B12 as cofactors were measured by this assay and used to reveal the binding properties of the cofactor by the Michaelis- Menten Method. The values of Km for coenzyme B12 in due to reconstitutions of holoenzyme in 2, 7 and 14 S: E were determined as; 1.12 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B12; 1.07 ± 0.04 μM and 0.35 ± 0.05 μM as obtained from respective 2 and 14 S: E compositions of holoenzyme. Analysis of these kinetics results curiously associates the increasing affinity of cofactors to apoenzyme with increased amount of component S used in reconstituting holoenzyme from separate apoenzyme components and cofactor. Moreover, in these studies a new method for assaying the activity of glutamate mutase was developed, whereby glutamate mutase activity is measured via depletion of NADH (λmax = 340 nm, ?340 = 6.3 mM?1?cm?1) as determined by UV spectrophotometry after addition of (2S, 3S)-3-methylaspartate and pyruvate to a mixture of E2S2-B12 and two auxiliary holoenzymes system; pyridoxal-5-phosphate dependent glutamate-pyruvate aminotransferase and NADH dependent (R)-2-hydroxyglutarate dehydrogenase. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (S)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (R)-2-hydroxylglutarate dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2S, 3S)-3-methylaspartate in the reaction of glutamate mutase as Km= 7 ± 0.07 mM and kcat= 0.54 ± 0.6 s?1. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, Keq = [(S<span style="font-family:Ve

Enzymatic Biosynthesis of l-2-Aminobutyric Acid by Glutamate Mutase Coupled with l-Aspartate-β-decarboxylase Using l-Glutamate as the Sole Substrate

l-2-Aminobutyric acid (L-ABA) is a chiral precursor of several important drugs. l-Glutamate is an ideal material for the production of L-ABA through γ-decarboxylation; however, enzyme-mediated γ-de...

Photolytic Cleavage of Co-C Bond in Coenzyme B12-Dependent Glutamate Mutase.

Glutamate mutase (GLM) is a coenzyme B12-dependent enzyme that catalyzes the conversion of S-glutamate to (2 S,3 S)-3-methyl aspartate. The initial step in the catalytic process is the homolytic cleavage of the coenzyme's Co-C bond upon binding of a substrate. Alternatively, the Co-C bond can be cleaved using light. To investigate the photolytic cleavage of the Co-C bond in GLM, we applied a combined density functional theory/molecular mechanics (DFT/MM) and time-dependent-DFT/MM method to scrutinize the ground and the low-lying excited states. Potential energy surfaces (PESs) were generated as a function of axial bond lengths to describe the photodissociation mechanism. The S1 PES was characterized as the crossing of two electronic states, metal-to-ligand charge transfer (MLCT), and ligand field (LF). In GLM, radical pairs generate from the LF state. Two pathways, path A and path B, were identified as possible channels to connect the MLCT and LF electronic states. The S1 PES in GLM was compared with the S1 PES for coenzyme B12-dependent ethanolamine ammonia lyase as well as the isolated AdoCbl cofactor. Finally, the theoretical insights related to the photodissociation mechanism were compared with transient absorption spectroscopy, electron paramagnetic resonance, and resonance Raman spectroscopy.

Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB.

Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency k cat/K m, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration.

Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway

Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as Clostridium tetanomorphum. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from C. tetanomorphum, were recombinantly expressed in Escherichia coli. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the in vivo activity. These efforts led to efficient mesaconate production at a titer of 7.81g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96g/L directly from glucose. In summary, we have engineered an efficient system in E. coli for the biosynthesis of mesaconate.

Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5′-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

Synthesis, solution and crystal structure of the coenzyme B12 analogue Coβ-2′-fluoro-2′,5′-dideoxyadenosylcobalamin

Crystal structure analyses have helped to decipher the mode of binding of coenzyme B12 (AdoCbl) in the active site of AdoCbl-dependent enzymes. However, the question of how such enzymes perform their radical reactions is still incompletely answered. A pioneering study by Gruber and Kratky of AdoCbl-dependent glutamate mutase (GLM) laid out a path for the movement of the catalytically active 5′-deoxyadenosyl radical, in which H-bonds between the protein and the 2′- and 3′-OH groups of the protein bound AdoCbl would play a decisive role. Studies with correspondingly modified coenzyme B12-analogues are of interest to gain insights into cofactor binding and enzyme mechanism. Here we report the preparation of Coβ-2′-fluoro-2′,5′-dideoxyadenosylcobalamin (2′FAdoCbl), which lacks the 2′-OH group critical for the interaction in enzymes. 2′FAdoCbl was prepared by alkylation of cob(I)alamin, obtained from the electrochemical reduction of aquocobalamin. Spectroscopic data and a single crystal X-ray analysis of 2′FAdoCbl established its structure, which was very similar to that one of coenzyme B12. 2′FAdoCbl is a 19F NMR active mimic of coenzyme B12 that may help to gain insights into binding interactions of coenzyme B12 with AdoCbl-dependent enzymes, proteins of B12 transport and of AdoCbl-biosynthesis, as well as with B12-riboswitches.

Production of vitamin B12 in recombinant Escherichia coli: an important step for heterologous production of structurally complex small molecules.

See accompanying article by Yeounjoo Ko et al. DOI 10.1002/biot.201400221 Vitamin B12 is one of the most complex small molecules acting as a co-factor in processes that are crucial to many living organisms. Vitamin B12 can be produced by fermentation of native bacterial producers with up to 10000 kilogram commercial production of vitamin B12 annually [1]. Because native bacterial producers are not the usual industrial producer strains, many attempts have been made to heterologously produce vitamin B12 in non-native microbial hosts. Since the de novo biosynthesis of vitamin B12 involves 30 enzyme-mediated reactions, those efforts have been unsuccessful until the recent study made by Ko et al. [2], which is published in this issue of Biotechnology Journal. The structure of vitamin B12 is so complex that Dorothy Crowfoot Hodgkin, who resolved the three-dimensional structure of vitamin B12 (cyanocobalamin) [3] and other important biochemical substances, was awarded the Nobel Prize in Chemistry in 1964. Chemical synthesis of vitamin B12 is a highly complicated process involving at least 60 steps, which is technically challenging and economically unviable. In nature, vitamin B12 is unique in the sense that its de novo biosynthesis appears to be limited to some bacteria and archaea [1]. This includes aerobi biosynthesis in Rhodobacter sphaeroides and Pseudomonas denitrificans, and anaerobic biosynthesis in Bacillus megaterium, Propionibacterium shermanii, Salmonella typhimurium and Lactobacillus reuteri [4]. Transferring a metabolic pathway from its native producer into heterologous microbial hosts has become a common practice in metabolic engineering. Nevertheless, assembling a functional metabolic pathway comprising more than 30 biochemical reactions remains a great challenge. Successful assembly of such a complex metabolic pathway requires that all of the genes be cloned accurately and expressed correctly, and that all of the introduced enzymes be functional so that the carbon flux can be transferred to the final target product. Very often, a single nucleotide mutation would result in failure of the entire pathway, and trouble-shooting would be very difficult and time-consuming. Ko et al. [2] use three compatible plasmids to clone the Pseudomonas denitrificans vitamin B12 pathway, which is encoded by 25 genes located in six different operons [2]. This strategy successfully resulted in the production of vitamin B12 in Escherichia coli, demonstrating that vitamin B12, which has the most complex structure amongst all vitamins, can be produced heterologously. Interestingly, although the synthesis of vitamin B12 in P. denitrificans is strictly oxygen-dependent, Ko et al. [2] show that the recombinant E. coli can produce vitamin B12 under anaerobic conditions. This suggests that the biosynthetic route towards vitamin B12 may not be dependent on molecular oxygen; the oxygen-dependant vitamin B12 biosynthesis in P. denitrificans might be due to some regulatory mechanisms in those native hosts, which is only effective under aerobic conditions. Biosynthesis of vitamin B12 appears to be restricted to a few representative bacteria and archaea, while vitamin B12-dependent enzymes are widespread throughout all domains of life [5]. Generally, vitamin B12-dependent reactions are described to catalyze methyl transfers and carbon backbone rearrangements. Besides the well-known diol and glycerol dehydratase, some additional examples of enzymes that require vitamin B12 include methionine synthase, methylmalonyl-CoA mutase, glutamate mutase, isobutyryl-CoA mutase, and reductive dehalogenases. For those organisms that do not possess vitamin B12 biosynthetic ability, addition of vitamin B12 to the medium is a necessity to initiate vitamin B12-dependent reactions. Production of vitamin B12 in recombinant E. coli enables scientists to consider vitamin B12-dependent biochemical reactions when designing new pathways, thus expanding the availability of enzymes. Previously, production of vitamin B12 in its native producers can be improved by optimization of the culture medium and process, mutagenesis of the producing strain, overexpression of the gene cluster involved in biosynthesis of vitamin B12, or optimizing promoters, ribosomal-binding sites and terminators [1]. Production of vitamin B12 in recombinant E. coli opens new possibilities for further improvement. Recently, engineered E. coli strains which are capable of producing some amino acids, organic acids, and alcohols have outcompeted their native producers. This demonstrates the power of applying synthetic biology approaches in strain improvement. With this rapid progress in E. coli cell factories, it can be optimistically predicted that production of vitamin B12 by engineered E. coli will have a bright future and lead to many more complex applications of metabolic engineering and synthetic biology.

Role of active site residues in promoting cobalt-carbon bond homolysis in adenosylcobalamin-dependent mutases revealed through experiment and computation.

Adenosylcobalamin (AdoCbl) serves as a source of reactive free radicals that are generated by homolytic scission of the coenzyme's cobalt-carbon bond. AdoCbl-dependent enzymes accelerate AdoCbl homolysis by ∼10(12)-fold, but the mechanism by which this is accomplished remains unclear. We have combined experimental and computational approaches to gain molecular-level insight into this process for glutamate mutase. Two residues, glutamate 330 and lysine 326, form hydrogen bonds with the adenosyl group of the coenzyme. A series of mutations that impair the enzyme's ability to catalyze coenzyme homolysis and tritium exchange with the substrate by 2-4 orders of magnitude were introduced at these positions. These mutations, together with the wild-type enzyme, were also characterized in silico by molecular dynamics simulations of the enzyme-AdoCbl-substrate complex with AdoCbl modeled in the associated (Co-C bond formed) or dissociated [adenosyl radical with cob(II)alamin] state. The simulations reveal that the number of hydrogen bonds between the adenosyl group and the protein side chains increases in the homolytically dissociated state, with respect to the associated state, for both the wild-type and mutant enzymes. The mutations also cause a progressive increase in the mean distance between the 5'-carbon of the adenosyl radical and the abstractable hydrogen of the substrate. Interestingly, the distance between the 5'-carbon and substrate hydrogen, determined computationally, was found to inversely correlate with the log k for tritium exchange (r = 0.93) determined experimentally. Taken together, these results point to a dual role for these residues: they both stabilize the homolytic state through electrostatic interactions between the protein and the dissociated coenzyme and correctly position the adenosyl radical to facilitate the abstraction of hydrogen from the substrate.

Molecular Docking Study on the Interaction of Riboflavin (Vitamin B2) and Cyanocobalamin (Vitamin B12) Coenzymes

Cobalamins are the largest and structurally complex cofactors found in biological systems and have attracted considerable attention due to their participation in the metabolic reactions taking place in humans, animals, and microorganisms. Riboflavin (vitamin B2) is a micronutrient and is the precursor of coenzymes, FMN and FAD, required for a wide variety of cellular processes with a key role in energy-based metabolic reactions. As coenzymes of both vitamins are the part of enzyme systems, the possibility of their mutual interaction in the body cannot be overruled. A molecular docking study was conducted on riboflavin molecule with B12 coenzymes present in the enzymes glutamate mutase, diol dehydratase, and methionine synthase by using ArgusLab 4.0.1 software to understand the possible mode of interaction between these vitamins. The results from ArgusLab showed the best binding affinity of riboflavin with the enzyme glutamate mutase for which the calculated least binding energy has been found to be −7.13 kcal/mol. The results indicate a significant inhibitory effect of riboflavin on the catalysis of B12-dependent enzymes. This information can be utilized to design potent therapeutic drugs having structural similarity to that of riboflavin.

Structural Basis of the Interaction of MbtH-like Proteins, Putative Regulators of Nonribosomal Peptide Biosynthesis, with Adenylating Enzymes

The biosynthesis of nonribosomally formed peptides (NRPs), which include important antibiotics such as vancomycin, requires the activation of amino acids through adenylate formation. The biosynthetic gene clusters of NRPs frequently contain genes for small, so-called MbtH-like proteins. Recently, it was discovered that these MbtH-like proteins are required for some of the adenylation reactions in NRP biosynthesis, but the mechanism of their interaction with the adenylating enzymes has remained unknown. In this study, we determined the structure of SlgN1, a 3-methylaspartate-adenylating enzyme involved in the biosynthesis of the hybrid polyketide/NRP antibiotic streptolydigin. SlgN1 contains an MbtH-like domain at its N terminus, and our analysis defines the parameters required for an interaction between MbtH-like domains and an adenylating enzyme. Highly conserved tryptophan residues of the MbtH-like domain critically contribute to this interaction. Trp-25 and Trp-35 form a cleft on the surface of the MbtH-like domain, which accommodates the alanine side chain of Ala-433 of the adenylating domain. Mutation of Ala-433 to glutamate abolished the activity of SlgN1. Mutation of Ser-23 of the MbtH-like domain to tyrosine resulted in strongly reduced activity. However, the activity of this S23Y mutant could be completely restored by addition of the intact MbtH-like protein CloY from another organism. This suggests that the interface found in the structure of SlgN1 is the genuine interface between MbtH-like proteins and adenylating enzymes.

Role of Tunneling in the Enzyme Glutamate Mutase

The role of quantum mechanical atom tunneling during the conversion of glutamate to methylaspartate catalyzed by glutamate mutase is investigated by quantum mechanical/molecular mechanical (QM/MM) simulations based on coupled cluster and density functional calculations. The use of instanton theory allows us to calculate the tunneling contributions of up to 78 atoms in the active site. We calculate kinetic isotope effects (KIEs) and compare them to experimental data. The simulations lead to deuterium KIEs of 10 for the hydrogen abstraction from glutamate substrate and 16 for the hydrogen abstraction from methylaspartate substrate, which are consistent with the experimental results. The hydrogen abstraction from methylaspartate has higher primary deuterium and tritium (46.1) KIEs than the abstraction from glutamate. The tunneling effect increases the reaction rate by a factor of 12.3 for the hydrogen abstraction from methylaspartate at 0. Tunneling is supported by the environment by preparing the enzyme through classical motions. Consideraton of the tunneling contributions of more and more atoms around the active center shows that the motions at the ribose ring play a central role during the tunneling enhancement of the hydrogen transfers. Our simulations give new insight into the catalytic process in glutamate mutase and the way enzymes use tunneling effects for a successful catalysis.