•5-Halokynurenines are good substrates for kynurenine monooxygenase.•3-Substituted kynurenines are competitive inhibitors of kynurenine monooxygenase.•A pharmacophore model was prepared and predicted 3,4-dichlorohippuric acid as an inhibitor.Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington’s and Alzheimer’s diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of kcat and kcat/Km comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with KI values lower than the Km for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a KI value of 1.5 μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The KI for this compound was found to be 34 μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO.Download high-res image (58KB)Download full-size image

•STM2360 encodes a d-ornithine/d-lysine decarboxylase.•The enzyme exhibits cooperativity, with a Hill coefficient ∼3.•STM2358 in the same operon is an ornithine racemase.•These enzymes are widely distributed in bacteria.STM2360 is a gene located in a small operon of undetermined function in Salmonella enterica serovar Typhimurium LT2. The amino acid sequence of STM2360 shows significant similarity (∼30% identity) to diaminopimelate decarboxylase (DapDC), a Fold III pyridoxal-5′-phosphate (PLP) dependent enzyme involved in l-lysine biosynthesis. We have found that the protein coded by STM2360 has a previously undocumented catalytic activity, d-ornithine/d-lysine decarboxylase (DOKDC). The reaction products, cadaverine and putrescine, respectively, were identified by NMR and mass spectrometry. The substrate specificity of DOKDC is d-Lysine > d-Ornithine. This is the first pyridoxal-5′-phosphate dependent decarboxylase identified to act on d-amino acids. STM2358, located in the same operon, has ornithine racemase activity. This suggests that the physiological substrate of the decarboxylase and the operon is ornithine. Homologs of STM2360 with high sequence identity (>80%) are found in other common enterobacteria, including species of Klebsiella, Citrobacter, Vibrio and Hafnia, as well as Clostridium in the Firmicutes, and Pseudomonas.Download high-res image (150KB)Download full-size image

•Mutant SADHs are useful as biocatalysts for ketone reduction.•Met-151 and Thr-153 are in the active site of SADH.•T153A and M151A mutations combined with I86A/C295A SADH change substrate specificity.Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus reduces ketones to chiral alcohols, and generally obeys Prelog's Rule, with binding pockets for large and small alkyl substituents, giving (S)-alcohols. We have previously shown that mutations in both the large and small pockets can alter both substrate specificity and stereoselectivity. In the present work, Met-151 and Thr-153, residues located in the small pocket, were mutated to alanine. The M151A mutant SADH shows significantly lower activity and lower stereoselectivity for reduction of aliphatic ketones than wild-type SADH. Furthermore, M151A showed non-linear kinetics for reduction of acetone. T153A SADH shows lower activity but similar stereoselectivity for ketone reduction compared to wild-type SADH. The I86A/M151A/C295A and I86A/T153A/C295A triple mutant SADH show altered specificity for reduction of substituted acetophenones. These results confirm that these mutations are useful to combine with I86A/C295A SADH to expand the small pocket of SADH and broaden the substrate specificity.Download high-res image (161KB)Download full-size image

The role of transition-state stabilization in enzyme catalysis, as proposed by Pauling, has been clearly demonstrated by extensive studies. In contrast, ground-state destabilization can also contribute to enzyme catalysis, but experimental evidence has been more limited. In recent years, high-resolution X-ray crystal structures of enzyme–substrate complexes have been obtained which show evidence for ground-state strain. We found that Y71F and F448H mutant tyrosine phenol-lyase (TPL) form complexes with 3-fluoro-l-tyrosine, a substrate, which shows a bending of the substrate aromatic ring about 20° out of plane, and we suggested that this was evidence for ground-state destabilization in TPL catalysis. Here, we have now evaluated quantitatively the role of ground-state destabilization in TPL catalysis. Phe-448 and Phe-449 are in close contact with the bound substrate side chain, and by mutating these residues to alanine and leucine, the contribution they play via ground-state destabilization was investigated. F448A, F448L and F449A TPL have activity for elimination of phenol from l-tyrosine reduced by a factor of 104, 103, and 104, respectively, but they have near-normal activity with the alternate substrates S-(o-nitrophenyl)-l-cysteine and S-ethyl-l-cysteine. F448A TPL forms quinonoid intermediates from l-tyrosine and S-ethyl-l-cysteine with rate constants similar to those of wild-type TPL. In addition, F448A TPL can form an aminoacrylate intermediate from S-ethyl-l-cysteine but not l-tyrosine, with a rate constant similar to that of wild-type TPL. Thus, the effect of the mutation is specifically on the elimination of phenol from l-tyrosine. We also examined the effect of hydrostatic pressure on the rates and equilibria of formation of the quinonoid intermediates from F448H and F448A TPL and 3-fluoro-l-tyrosine. Although the fastest phase shows only a small effect of pressure, the three slower phases have significant pressure dependences, suggesting that they may be associated with a conformational change. These results demonstrate that Phe-448 and Phe-449 contribute a total of about 108 to catalysis in TPL, about 50% of the estimated rate acceleration, by introducing ground-state destabilization into the l-tyrosine substrate.Keywords: enzyme mechanism; ground-state strain; kinetics; pyridoxal-5′-phosphate; rate acceleration

We have designed, synthesized, and evaluated tyrosine homologues and their O-methyl derivatives as potential inhibitors for tyrosine phenol lyase (TPL, E.C. 4.1.99.2). Recently, we reported that homologues of tryptophan are potent inhibitors of tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1), with Ki values in the low µM range (Do et al. Arch Biochem Biophys 560:20–26, 2014). As the structure and mechanism for TPL is very similar to that of TIL, we postulated that tyrosine homologues could also be potent inhibitors of TPL. However, we have found that homotyrosine, bishomotyrosine, and their corresponding O-methyl derivatives are competitive inhibitors of TPL, which exhibit Ki values in the range of 0.8–1.5 mM. Thus, these compounds are not potent inhibitors, but instead bind with affinities similar to common amino acids, such as phenylalanine or methionine. Pre-steady-state kinetic data were very similar for all compounds tested and demonstrated the formation of an equilibrating mixture of aldimine and quinonoid intermediates upon binding. Interestingly, we also observed a blue-shift for the absorbance peak of external aldimine complexes of all tyrosine homologues, suggesting possible strain at the active site due to accommodating the elongated side chains.

The ability to control the substrate specificity and stereochemistry of enzymatic reactions is of increasing interest in biocatalysis. As this review highlights, this control can be achieved through various means, including mutagenesis of active site residues and alteration of physical variables such as temperature and pressure as well as through changing the reaction medium. Although the focus of this article is on alcohol dehydrogenase reactions, each of these techniques can be readily applied toward other enzyme classes, as well.Keywords: alcohol dehydrogenase; medium engineering; mutagenesis; protein engineering; stereospecificity; substrate specificity

The effects of hydrostatic pressure and temperature on the reaction of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus with enantiomers of 2-butanol, 2-pentanol, and 2-hexanol have been measured. For all substrates, increasing pressure favors the S enantiomer, whereas increasing temperature favors the R enantiomer. Fitting of the pressure and temperature data for 2-hexanol provided apparent ΔΔS‡ and ΔΔV‡ values of +46 ± 15 J/mol and +(2.0 ± 0.4) × 10–2 L/mol, respectively. These results support our previous proposal that desolvation of the enzyme active site plays an important role in stereospecificity.Keywords: alcohol dehydrogenase; hydrostatic pressure; NADP; secondary alcohol; stereochemistry; temperature

Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.

•TPL and TIL catalyze the elimination reactions of l-Tyr and l-Trp.•TPL and TIL have similar three dimensional structures and high sequence homology.•Both enzymes use acid–base catalysis in the reaction mechanism.•X-ray crystal structures of mutant TPL show that the bound substrate is strained.•TPL and TIL use both acid–base catalysis and substrate strain for catalysis.The carbon–carbon lyases, tryptophan indole lyase (TIL) and tyrosine phenol-lyase (TPL) are bacterial enzymes which catalyze the reversible elimination of indole and phenol from l-tryptophan and l-tyrosine, respectively. These PLP-dependent enzymes show high sequence homology (∼40% identity) and both form homotetrameric structures. Steady state kinetic studies with both enzymes show that an active site base is essential for activity, and α-deuterated substrates exhibit modest primary isotope effects on kcat and kcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500 nm. In the presence of phenol and indole analogues, 4-hydroxypyridine and benzimidazole, the quinonoid intermediates of TPL and TIL decay to aminoacrylate intermediates, with λmax at about 340 nm. Surprisingly, there are significant kinetic isotope effects on both formation and subsequent decay of the quinonoid intermediates when α-deuterated substrates are used. The crystal structure of TPL with a bound competitive inhibitor, 4-hydroxyphenylpropionate, identified several essential catalytic residues: Tyr-71, Thr-124, Arg-381, and Phe-448. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg-381(TPL)/Ile-396 (TIL); Thr-124 (TPL)/Asp-137 (TIL), and Phe-448 (TPL)/His-463 (TIL). Mutagenesis of these residues results in dramatic decreases in catalytic activity without changing substrate specificity. The conserved tyrosine, Tyr-71 (TPL)/Tyr-74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group. Mutation of Arg-381 and Thr-124 of TPL to alanine results in very low but measurable catalytic activity. Crystallography of Y71F and F448H TPL with 3-fluoro-l-tyrosine bound demonstrated that there are two quinonoid structures, relaxed and tense. In the relaxed structure, the substrate aromatic ring is in plane with the Cβ–Cγ bond, but in the tense structure, the substrate aromatic ring is about 20° out of plane with the Cβ–Cγ bond. In the tense structure, hydrogen bonds are formed between the substrate OH and the guanidinium of Arg-381 and the OH of Thr-124, and the phenyl rings of Phe-448 and 449 provide steric strain. Based on the effects of mutagenesis, the substrate strain is estimated to contribute about 108 to TPL catalysis. Thus, the mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL.

A series of substituted kynurenines (3-bromo-dl, 3-chloro-dl, 3-fluoro-dl, 3-methyl-dl, 5-bromo-l, 5-chloro-l, 3,5-dibromo-l and 5-bromo-3-chloro-dl) have been synthesized and tested for their substrate activity with human and Pseudomonas fluorescens kynureninase. All of the substituted kynurenines examined have substrate activity with both human as well as P. fluorescens kynureninase. For the human enzyme, 3- and 5-substituted kynurenines have kcat and kcat/Km values higher than l-kynurenine, but less than that of the physiological substrate, 3-hydroxykynurenine. However, 3,5-dibromo- and 5-bromo-3-chlorokynurenine have kcat and kcat/Km values close to that of 3-hydroxykynurenine with human kynureninase. The effects of the 3-halo substituents on the reactivity with human kynureninase may be due to electronic effects and/or halogen bonding. In contrast, for the bacterial enzyme, 3-methyl, 3-halo and 3,5-dihalokynurenines are much poorer substrates, while 3-fluoro, 5-bromo, and 5-chlorokynurenine have kcat and kcat/Km values comparable to that of its physiological substrate, l-kynurenine. Thus, 5-bromo and 5-chloro-l-kynurenine are good substrates for both human as well as bacterial enzyme, indicating that both enzymes have space for substituents in the active site near C-5. The increased activity of the 5-halokynurenines may be due to van der Waals contacts or hydrophobic effects. These results may be useful in the design of potent and/or selective inhibitors of human and bacterial kynureninase.

Osmolytes are common constituents of bacteria that may be produced or accumulate at high concentrations, up to 1 M, when cells are subjected to stresses like ionic strength and temperature. However, the effects of osmolytes on the allosteric properties of bacterial enzymes have rarely been examined. We have studied the effects of osmolytes and hydrostatic pressure on the allosteric equilibria of Salmonella typhimurium tryptophan (Trp) synthase. Trp synthase is a well-studied multienzyme complex with activity tightly regulated by allosteric interactions between the α- and β-subunits. Trp synthase activity is affected by a wide range of physical parameters, including monovalent cations, pH, ligands, solvents, and hydrostatic pressure. Osmolytes, including betaine, taurine, sucrose, and polyethylene glycol, activate Trp synthase 2–3-fold in the absence of monovalent cations, indicating that osmolytes can stabilize the active closed conformation. However, in the presence of monovalent cations, osmolytes have only minor effects on activity and allosteric equilibria, but 1 M betaine stabilizes the Trp synthase–Ser–indoline complex against apparent pressure-induced subunit dissociation. Na+ and K+ are more effective at shifting the α-aminoacrylate–indoline quinonoid equilibrium toward the quinonoid side, with a KQ of 8–10, than NH4+(KQ ∼ 2). Furthermore, pressure-jump experiments show that the mechanism of indoline reaction to form a quinonoid complex may be different for the NH4+ enzyme than the Na+ and K+ forms. These results show that osmolytes have subtle but significant effects on the allosteric properties of Trp synthase, and these effects may be important in vivo.

The effects of pH and hydrostatic pressure on the reaction of H463F tryptophan indole-lyase (TIL) have been evaluated. The mutant TIL shows very low activity for elimination of indole but is still competent to form a quinonoid intermediate from l-tryptophan [Phillips, R. S., Johnson, N., and Kamath, A. V. (2002) Biochemistry 41, 4012–4019]. Stopped-flow measurements show that the formation of the quinonoid intermediate at 505 nm is affected by pH, with a bell-shaped dependence for the forward rate constant, kf, and dependence on a single basic group for the reverse rate constant, kr, with the following values: pKa1 = 8.14 ± 0.15, pKa2 = 7.54 ± 0.15, kf,min = 18.1 ± 1.3 s–1, kf,max = 179 ± 46.3 s–1, kr,min = 11.4 ± 1.2 s–1, and kr,max = 33 ± 1.6 s–1. The pH effects may be due to ionization of Tyr74 as the base and Cys298 as the acid influencing the rate constant for deprotonation. High-pressure stopped-flow measurements were performed at pH 8, which is the optimum for the forward reaction. The rate constants show an increase with pressure up to 100 MPa and a subsequent decrease above 100 MPa. Fitting the pressure data gives the following values: kf,0 = 15.4 ± 0.8 s–1, ΔV⧧ = −29.4 ± 2.9 cm3 mol–1, and Δβ⧧ = −0.23 ± 0.03 cm3 mol–1 MPa–1 for the forward reaction, and kr,0 = 20.7 ± 0.8 s–1, ΔV⧧ = −9.6 ± 2.3 cm3 mol–1, and Δβ⧧ = −0.05 ± 0.02 cm3 mol–1 MPa–1 for the reverse reaction. The primary kinetic isotope effect on quinonoid intermediate formation at pH 8 is small (∼2) and is not significantly pressure-dependent, suggesting that the effect of pressure on kf may be due to perturbation of an active site preorganization step. The negative activation volume is also consistent with preorganization of the ES complex prior to quinonoid intermediate formation, and the negative compressibility may be due to the effect of pressure on the enzyme conformation. These results support the conclusion that the preorganization of the H463F TIL Trp complex, which is probably dominated by motion of the l-Trp indole moiety of the aldimine complex, contributes to quinonoid intermediate formation.

•Homologues of l-tryptophan, l-homotryptophan and l-bishomotryptophan, were prepared.•These homologues inhibit tryptophan indole-lyase, but not tryptophan synthase.•l-Bishomotryptophan is a more potent inhibitor than l-homotryptophan.•l-Homotryptophan forms a quinonoid complex with tryptophan indole-lyase.•l-Bishomotryptophan does not form a quinonoid complex with tryptophan indole-lyase.We have designed, synthesized and evaluated homotryptophan analogues as possible mechanism-based inhibitors for Escherichia coli tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1). As a quinonoid structure is an intermediate in the reaction mechanism of TIL, we anticipated that homologation of the physiological substrate, l-Trp would provide analogues resembling the transition state for β-elimination, and potentially inhibit TIL. Our results demonstrate that l-homotryptophan (1a) is a moderate competitive inhibitor of TIL, with Ki = 67 μM, whereas l-bishomotryptophan (1b) displays more potent inhibition, with Ki = 4.7 μM. Pre-steady-state kinetics indicated the formation of an external aldimine and quinonoid with 1a, but only the formation of an external aldimine for 1b, suggesting differences in the inhibition mechanism. These results demonstrate that formation of a quinonoid complex is not required for strong inhibition. In addition, the Trp analogues were evaluated as inhibitors of Salmonella typhimurium Trp synthase. Our results indicate that compound 1b is at least 25-fold more selective toward TIL than Trp synthase. We report that compound 1b is comparable to the most potent inhibitor previously reported, while displaying high selectivity for TIL. Thus, 1b is a potential lead for the development of novel antibacterials.

Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.