Biomedical subjects

Michael D Toney

Publications and source records attributed to Michael D Toney.

At least 19 recordsLinked to original sources

PubMed2006

Aminodeoxychorismate synthase inhibitors from one-bead one-compound combinatorial libraries: "staged" inhibitor design.

4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in the conversion of chorismate into p-aminobenzoate, which is incorporated into folic acid. We aim to discover compounds that inhibit ADCS and serve as leads for a new class of antimicrobial compounds. This report presents (1) synthesis of a mass-tag encoded library based on a "staged" design, (2) massively parallel fluorescence-based on-bead screening, (3) rapid structural identification of hits, and (4) full kinetic analysis of ADCS. All inhibitors are competitive against chorismate and Mg(2+). The most potent ADCS inhibitor identified has a K(i) of 360 microM. We show that the combinatorial diversity elements add substantial binding affinity by interacting with residues outside of but proximal to the active site. The methods presented here constitute a paradigm for inhibitor discovery through active site targeting, enabled by rapid library synthesis, facile massively parallel screening, and straightforward hit identification.

Acetates↗

PubMed2006

X-ray crystallographic structures of enamine and amine Schiff bases of pyridoxal and its 1:1 hydrogen-bonded complexes with benzoic acid derivatives: evidence for coupled inter- and intramolecular proton transfer.

Crystal structures of Schiff bases containing pyridoxal (PL), N-(pyridoxylidene)-tolylamine, C(15)H(16)N(2)O(2) (I), N-(pyridoxylidene)-methylamine, C(9)H(12)N(2)O(2) (III), and their 1:1 adduct with 2-nitrobenzoic acid, (I)(+) C(7)H(4)NO_4;- (II), and 4-nitrobenzoic acid, (III)(+) C(7)H(4)NO_4;- (IV), serve as models for the coenzyme pyridoxal-5'-phosphate (PLP) in its PLP-dependent enzymes. These models allow the study of the intramolecular OHN hydrogen bond of PL/PLP Schiff bases and the H-acceptor properties of their pyridine rings. The free base (I) forms hydrogen-bonded chains involving the hydroxyl side groups and the rings of adjacent molecules, whereas (III) forms related hydrogen-bonded cyclic dimers. The adducts (II)/(IV) consist of 1:1 hydrogen-bonded complexes, exhibiting strong intermolecular bonds between the carboxylic groups of the acids and the pyridine rings of (I)/(III). In conclusion, the proton in the intramolecular O-H...N hydrogen bond of (I)/(III) is located close to oxygen (enolamine form). The added acids protonate the pyridine ring in (II)/(IV), but only in the latter case does this protonation lead to a shift of the intramolecular proton towards the nitrogen (ketoimine form). All crystallographic structures were observed in the open form. In contrast, the formation of the pyridinium salt by dissolving (IV) leads to the cyclic aminal form.

Benzoates↗

PubMed2006

Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries.

One-bead one-compound combinatorial chemistry together with a high-throughput screen based on fluorescently labeled enzyme allowed the identification of slow binding inhibitors of human serine racemase (hSR). A peptide library of topographically segregated encoded resin beads was synthesized, and several hSR-binding compounds were isolated, identified, and resynthesized for further kinetic study. Of these, several showed inhibitory effects with moderate potency (high micromolar K(I)s) toward hSR. A clear structural motif was identified consisting of 3-phenylpropionic acid and histidine moieties. Importantly, the inhibitors identified showed no structural similarities to the natural substrate, L-serine. Detailed kinetic analyses of the properties of selected inhibitors show that the screening protocol used here selectively identifies slow binding inhibitors. They provide a pharmacophore for the future isolation of more potent ligands that may prove useful in probing and understanding the biological role of hSR.

Combinatorial Chemistry Techniques↗

PubMed2006

Direct detection and kinetic analysis of covalent intermediate formation in the 4-amino-4-deoxychorismate synthase catalyzed reaction.

Chorismate-utilizing enzymes catalyze diverse reactions, providing critical physiological functions unique to plants, bacteria, fungi, and some parasites. Their absence in animals makes them excellent targets for antimicrobials and herbicides. 4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in folate biosynthsis and shares a common core mechanism with isochorismate synthase (IS) and anthranilate synthase (AS), in which nucleophile addition at C2 initiates these reactions. Evidence was presented previously [He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-2385] that K274 is the nucleophile in ADCS, implying formation of a covalent intermediate. Herein, we report the direct detection of this covalent intermediate formed in ADCS-catalyzed reactions by ESI-MS. Difference spectra show the covalent intermediate has an absorption maximum at 310 nm. This was used to study the pre-steady-state kinetics of covalent intermediate formation under various conditions. Additionally, E258 in ADCS was shown to be critical to formation of the covalent intermediate by acting as a general acid catalyst for loss of the C4 hydroxyl group. The E258A/D mutants both exhibit very low activity. Acetate is a poor chemical rescue agent for E258D but an excellent one for E258A, with a 20000-fold and 3000-fold rate increase for Gln-dependent and NH(4)(+)-dependent activities, respectively. Lastly, A213 in IS (structurally homologous to K274 in ADCS) was changed to lysine in an attempt to convert IS to an ADCS-like enzyme. HPLC studies support the formation of a covalent intermediate with this mutant.

Carbon-Nitrogen Ligases↗

PubMed2006

NMR studies of solvent-assisted proton transfer in a biologically relevant Schiff base: toward a distinction of geometric and equilibrium H-bond isotope effects.

The tautomeric equilibrium in a Schiff base, N-(3,5-dibromosalicylidene)-methylamine 1, a model for the hydrogen bonded structure of the cofactor pyridoxal-5'-phosphate PLP which is located in the active site of the enzyme, was measured by means of 1H and 15N NMR and deuterium isotope effects on 15N chemical shifts at variable temperature and in different organic solvents. The position of the equilibrium was estimated using the one-bond 1J(OHN) and vicinal 3J(H(alpha)CNH) scalar coupling constants. Additionally, DFT calculations of a series of Schiff bases, N-(R1-salicylidene)-alkyl(R2)amines, were performed to obtain the hydrogen bond geometries. The latter made it possible to investigate a broad range of equilibrium positions. The increase of the polarity of the aprotic solvent shifts the proton in the intramolecular OHN hydrogen bond closer to the nitrogen. The addition of methanol and of hexafluoro-2-propanol to 1 in aprotic solvents models the PLP-water interaction in the enzymatic active site. The alcohols, which vary in acidity and change the polarity around the hydrogen bond, also stabilize the equilibrium, so that the proton is shifted to the nitrogen.

Aspartate Aminotransferases↗

PubMed2005

Role of Q52 in catalysis of decarboxylation and transamination in dialkylglycine decarboxylase.

Dialkylglycine decarboxylase (DGD) is a pyridoxal phosphate dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. DGD uses stereoelectronic effects to control its unusual reaction specificity. X-ray crystallographic structures of DGD suggest that Q52 is important in maintaining the substrate carboxylate in a stereoelectronically activated position. Here, the X-ray structures of the Q52A mutant and the wild type (WT) DGD-PMP enzymes are presented, as is the analysis of steady-state and half-reaction kinetics of three Q52 mutants (Q52A, Q52I, and Q52E). As expected if stereoelectronic effects are important to catalysis, the steady-state rate of decarboxylation for all three mutants has decreased significantly compared to that of WT. Q52A exhibits an approximately 85-fold decrease in k(cat) relative to that of WT. The rate of the decarboxylation half-reaction decreases approximately 10(5)-fold in Q52I and approximately 10(4)-fold in Q52E compared to that of WT. Transamination half-reaction kinetics show that Q52A and Q52I have greatly reduced rates compared to that of WT and are seriously impaired in pyridoxamine phosphate (PMP) binding, with K(PMP) at least 50-100-fold greater than that of WT. The larger effect on the rate of l-alanine transamination than of pyruvate transamination in these mutants suggests that the rate decrease is the result of selective destabilization of the PMP form of the enzyme in these mutants. Q52E exhibits near-WT rates for transamination of both pyruvate and l-alanine. Substrate binding has been greatly weakened in Q52E with apparent dissociation constants at least 100-fold greater than that of WT. The rate of decarboxylation in Q52E allows the energetic contribution of stereoelectronic effects, DeltaG(stereoelectronic), to be estimated to be -7.3 kcal/mol for DGD.

Amination↗

PubMed2005

Ionization state of pyridoxal 5'-phosphate in D-serine dehydratase, dialkylglycine decarboxylase and tyrosine phenol-lyase and the influence of monovalent cations as inferred by 31P NMR spectroscopy.

The 31P NMR spectroscopy of three pyridoxal 5'-phosphate-dependent enzymes, monomeric D-serine dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied. 31P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by approximately 3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The 31P NMR signal of the phosphate group of pyridoxal 5'-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. For D-serine dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from 31P NMR data.

Carboxy-Lyases↗

PubMed2005

Kinetic and crystallographic analysis of active site mutants of Escherichia coli gamma-aminobutyrate aminotransferase.

The E. coli isozyme of gamma-aminobutyrate aminotransferase (GABA-AT) is a tetrameric pyridoxal phosphate-dependent enzyme that catalyzes transamination between primary amines and alpha-keto acids. The roles of the active site residues V241, E211, and I50 in the GABA-AT mechanism have been probed by site-directed mutagenesis. The beta-branched side chain of V241 facilitates formation of external aldimine intermediates with primary amine substrates, while E211 provides charge compensation of R398 selectively in the primary amine half-reaction and I50 forms a hydrophobic lid at the top of the substrate binding site. The structures of the I50Q, V241A, and E211S mutants were solved by X-ray crystallography to resolutions of 2.1, 2.5, and 2.52 A, respectively. The structure of GABA-AT is similar in overall fold and active site structure to that of dialkylglycine decarboxylase, which catalyzes both transamination and decarboxylation half-reactions in its normal catalytic cycle. Therefore, an attempt was made to convert GABA-AT into a decarboxylation-dependent aminotransferase similar to dialkylglycine decarboxylase by systematic mutation of E. coli GABA-AT active site residues. Two of the twelve mutants presented, E211S/I50G/C77K and E211S/I50H/V80D, have approximately 10-fold higher decarboxylation activities than the wild-type enzyme, and the E211S/I50H/V80D has formally changed the reaction specificity to that of a decarboxylase.

4-Aminobutyrate Transaminase↗

PubMed2005

Reaction specificity in pyridoxal phosphate enzymes.

Pyridoxal phosphate enzymes catalyze a wide variety of reaction types on amines and amino acids, generally by stabilizing carbanionic intermediates. This makes them very useful in cellular metabolism, but it also creates problems in controlling the reaction pathway that a given enzyme follows, i.e., in controlling reaction specificity. Stereoelectronic effects have been proposed to play a major role in determining the bond to Calpha that gets broken in the external aldimine intermediate that is common to all PLP enzymes. Here, we discuss our work on dialkylglycine decarboxylase aimed at providing direct evidence for stereoelectronic control of external aldimine reactivity. Once a bond to Calpha has been broken to form the carbanionic intermediate, enzymes must also carefully control the fate of this reactive species. Our studies with alanine racemase suggest that the enzyme selectively destabilizes the carbanionic quinonoid intermediate to promote higher racemization specificity by avoiding transamination side reactions.

Alanine Racemase↗

PubMed2004

Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity.

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.

Adenosine Triphosphate↗

PubMed2004

Crystal structures of unbound and aminooxyacetate-bound Escherichia coli gamma-aminobutyrate aminotransferase.

The X-ray crystal structures of Escherichia coli gamma-aminobutyrate aminotransferase unbound and bound to the inhibitor aminooxyacetate are reported. The enzyme crystallizes from ammonium sulfate solutions in the P3(2)21 space group with a tetramer in the asymmetric unit. Diffraction data were collected to 2.4 A resolution for the unliganded enzyme and 1.9 A resolution for the aminooxyacetate complex. The overall structure of the enzyme is similar to those of other aminotransferase subgroup II enzymes. The ability of gamma-aminobutyrate aminotransferase to act on primary amine substrates (gamma-aminobutyrate) in the first half-reaction and alpha-amino acids in the second is proposed to be enabled by the presence of Glu211, whose side chain carboxylate alternates between interactions with Arg398 in the primary amine half-reaction and an alternative binding site in the alpha-amino acid half-reaction, in which Arg398 binds the substrate alpha-carboxylate. The specificity for a carboxylate group on the substrate side chain is due primarily to the presence of Arg141, but also requires substantial local main chain rearrangements relative to the structurally homologous enzyme dialkylglycine decarboxylase, which is specific for small alkyl side chains. No iron-sulfur cluster is found in the bacterial enzyme as was found in the pig enzyme [Storici, P., De Biase, D., Bossa, F., Bruno, S., Mozzarelli, A., Peneff, C., Silverman, R. B., and Schirmer, T. (2004) J. Biol. Chem. 279, 363-73.]. The binding of aminooxyacetate causes remarkably small changes in the active site structure, and no large domain movements are observed. Active site structure comparisons with pig gamma-aminobutyrate aminotransferase and dialkylglycine decarboxylase are discussed.

4-Aminobutyrate Transaminase↗

PubMed2004

Biliverdin reduction by cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) proceeds via linear tetrapyrrole radical intermediates.

Cyanobacterial phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the four electron reduction of biliverdin IXalpha (BV) to phycocyanobilin, a key step in the biosynthesis of the linear tetrapyrrole (bilin) prosthetic groups of cyanobacterial phytochromes and the light-harvesting phycobiliproteins. Using an anaerobic assay protocol, optically detected bilin-protein intermediates, produced during the PcyA catalytic cycle, were shown to correlate well with the appearance and decay of an isotropic g approximately 2 EPR signal measured at low temperature. Absorption spectral simulations of biliverdin XIIIalpha reduction support a mechanism involving direct electron transfers from ferredoxin to protonated bilin:PcyA complexes.

Biliverdine↗

PubMed2004

Alanine racemase free energy profiles from global analyses of progress curves.

Free energy profiles for alanine racemase from Bacillus stearothermophilus have been determined at pH 6.9 and 8.9 from global analysis of racemization progress curves. This required a careful statistical design due to the problems in finding the global minimum in mean square for a system with eight adjustable parameters (i.e., the eight rate constants that describe the stepwise chemical mechanism). The free energy profiles obtained through these procedures are supported by independent experimental evidence: (1). steady-state kinetic constants, (2). solvent viscosity dependence, (3). spectral analysis of reaction intermediates, (4). equilibrium overshoots for progress curves measured in D(2)O, and (5). the magnitudes of calculated intrinsic kinetic isotope effects. The free energy profiles for the enzyme are compared to those of the uncatalyzed and the PLP catalyzed reactions. At pH 6.9, PLP lowers the free energy of activation for deprotonation by 8.4 kcal/mol, while the inclusion of apoenzyme along with PLP additionally lowers it by 11 kcal/mol.

Alanine↗

PubMed2004

Kinetic and thermodynamic analysis of the interaction of cations with dialkylglycine decarboxylase.

Dialkylglycine decarboxylase (DGD) is a tetrameric pyridoxal phosphate (PLP)-dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. Its activity is dependent on cations. Metal-free DGD and DGD complexes with seven monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) and three divalent cations (Mg(2+), Ca(2+), and Ba(2+)) have been studied. The catalytic rate constants for cation-bound enzyme (ck(cat) and ck(cat)/bK(AIB)) are cation-size-dependent, K(+) being the monovalent cation with the optimal size for catalytic activity. The divalent alkaline earth cations (Mg(2+), Ca(2+), and Ba(2+)) all give approximately 10-fold lower activity compared to monovalent alkali cations of similar ionic radius. The Michaelis constant for aminoisobutyrate (AIB) binding to DGD-PLP complexes with cations (bK(AIB)) varies with ionic radius. The larger cations (K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) give smaller bK(AIB) ( approximately 4 mM), while smaller cations (Li(+), Na(+)) give larger values (approximately 10 mM). Cation size and charge dependence is also found with the dissociation constant for PLP binding to DGD-cation complexes (aK(PLP)). K(+) and Rb(+) possess the optimal ionic radius, giving the lowest values of aK(PLP). The divalent alkaline earth cations give aK(PLP) values approximately 10-fold higher than alkali cations of similar ionic radius. The cation dissociation constant for DGD-PLP-AIB-cation complexes (betaK(M)z+) was determined and also shown to be cation-size-dependent, K(+) and Rb(+) yielding the lowest values. The kinetics of PLP association and dissociation from metal-free DGD and its complexes with cations (Na(+), K(+), and Ba(2+)) were analyzed. All three cations tested increase PLP association and decrease PLP dissociation rate constants. Kinetic studies of cation binding show saturation kinetics for the association reaction. The half-life for association with saturating Rb(+) is approximately 24 s, while the half-life for dissociation of Rb(+) from the DGD-PLP-AIB-Rb(+) complex is approximately 12 min.

Binding Sites↗

PubMed2004

Conservation of mechanism in three chorismate-utilizing enzymes.

Chorismate is the end-product of the shikimate pathway for biosynthesis of carbocyclic aromatic compounds in plants, bacteria, fungi, and some parasites. Anthranilate synthase (AS), 4-amino-4-deoxychorismate synthase (ADCS), and isochorismate synthase (IS) are homologous enzymes that carry out the initial transformations on chorismate in the biosynthesis of tryptophan, p-aminobenzoate, and enterobactin, respectively, and are expected to share a common mechanism. Poor binding to ADCS of two potential transition state analogues for addition of a nucleophile to C6 of chorismate implies that it, like AS and IS, initiates reaction by addition of a nucleophile to C2. Molecular modeling based on the X-ray structures of AS and ADCS suggests that the active site residue K274 is the nucleophile employed by ADCS to initiate the reaction, forming a covalent intermediate. The K274A and K274R mutants were shown to have 265- and 640-fold reduced k(cat) values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH(4)(+), ADCS and the K274A mutant have identical k(cat) values, suggesting the participation of NH(4)(+) as a rescue agent. Such participation was confirmed by the buildup of 2-amino-2-deoxyisochorismate in the reactions of the K274A mutant but not ADCS, when either NH(4)(+) or PabA + glutamine is used as the nitrogen source. Additionally, the inclusion of ethylamine in the reactions of K274A yields the N-ethyl derivative of 2-amino-2-deoxyisochorismate. A unifying mechanism for AS, ADCS, and IS entailing nucleophile addition to C2 of chorismate in an S(N)2' ' process is proposed.

Anthranilate Synthase↗

PubMed2003

Kinetic analysis of the 4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyne antitumor antibiotic C-1027 biosynthesis.

The enediyne antitumor antibiotic C-1027 contains an unusual (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety, which requires an aminomutase for its biosynthesis. Previously, we established that SgcC4 is an aminomutase that catalyzes the conversion of L-tyrosine to (S)-beta-tyrosine and employs 4-methylideneimidazole-5-one (MIO) at its active site [Christenson, S. D., Liu, W., Toney, M. D., and Shen, B. (2003) J. Am. Chem. Soc. 125, 6062-6063]. Here, we present a thorough analysis of the properties of SgcC4. L-Tyrosine is the best substrate among those tested and most likely serves as the in vivo precursor for the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety. The presence of MIO in the active site is supported by several lines of evidence. (1) Addition of ATP or divalent metal ions has no effect on its aminomutase activity. (2) SgcC4 has optimal activity at pH approximately 8.8, similar to the pH optima of MIO-dependent ammonia lyases. (3) SgcC4 is strongly inhibited by sodium borohydride and potassium cyanide, but preincubation with L-tyrosine or 4-hydroxycinnamate largely prevents this inhibition. (4) The difference spectrum between SgcC4 and its S153A mutant shows a positive peak at approximately 310 nm, indicative of MIO. (5) The S153A mutation lowers k(cat)/K(M) 640-fold. The SgcC4-catalyzed conversion of L-tyrosine to (S)-beta-tyrosine proceeds via 4-hydroxycinnamate as an intermediate. The latter also acts as a competitive inhibitor with respect to L-tyrosine and serves as an alternative substrate for the production of beta-tyrosine in the presence of an amino source. A full time course for the SgcC4-catalyzed interconversion between L-tyrosine, beta-tyrosine, and 4-hydroxycinnamate was measured and analyzed to provide estimates for the rate constants in a minimal mechanism. SgcC4 also exhibits a beta-tyrosine racemase activity, but alpha-tyrosine racemase activity was not detected.

Amino Acid Sequence↗

PubMed2003

A continuous-flow system for high-precision kinetics using small volumes.

A generally applicable continuous-flow kinetic analysis system that gives data of a precision high enough to measure small kinetic isotope effects for enzymatic and nonenzymatic reactions is described. It employs commercially available components that are readily assembled into an apparatus that is easy to use. It operates under laminar flow conditions, which requires that the time between the initiation of the reaction in the mixer and the observation be long enough that molecular diffusion can effect a symmetrization of the concentration profile that results from a thin plug of reagents introduced at the mixer. The analysis of a second-order irreversible reaction under pseudo-first-order conditions is presented. The Yersinia pestis protein tyrosine phosphatase catalyzed hydrolysis of p-nitrophenyl phosphate is characterized with the system, and a proton inventory on kcat is presented.

Diffusion↗

PubMed2003

A novel 4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyne antitumor antibiotic C-1027 biosynthesis.

The C-1027 enediyne antibiotic contains an unusual 3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is thought to be derived from tyrosine by an aminomutase reaction. However, none of the genes identified within the C-1027 gene cluster encode proteins with strong homology to known aminomutases. The sgcC4 gene encodes a protein with strong homology to dehydroalanine-dependent histidine/phenylalanine ammonia lyases. The sgcC4 gene was expressed in E. coli, and overproduced SgcC4 was purified as a His6-tagged fusion protein. Biochemical characterization of the purified SgcC4 establishes that SgcC4 is an aminomutase that catalyzes the conversion of l-tyrosine to (S)-beta-tyrosine and employs 4-methylideneimidazole-5-one (MIO) at its active site. The latter was supported by borohydride and cyanide inhibition studies and confirmed by site-directed mutagenesis. The S153A mutant exhibited a 340-fold decrease in kcat/KM. SgcC4 represents a novel type of aminomutase, extending the known MIO chemistry from ammonia lyases into aminomutases.

Alanine↗