External Aldimine Complex Research Articles

The Y430F mutant of Salmonella d-ornithine/d-lysine decarboxylase has altered stereospecificity and a putrescine allosteric activation site

Tyrosine-430 of d-ornithine/d-lysine decarboxylase (DOKDC) is located in the active site, and was suggested to be responsible for the D-stereospecificity of the enzyme. We have prepared the Y430F mutant form of Salmonella enterica serovar typhimurium DOKDC and evaluated its catalytic activity with D- and l-lysine and ornithine. The kinetic results show that the Y430F mutant has measurable decarboxylase activity with both D- and l-lysine and ornithine, which wild type DOKDC does not. Spectroscopic experiments show that these amino acids bind to form external aldimine complexes with the pyridoxal-5′-phosphate with λmax = 425 nm. In addition, we have obtained crystal structures of Y430F DOKDC bound to HEPES, putrescine, d-ornithine, d-lysine, and d-arginine. The d-amino acids bind in the crystals to form equilibrium mixtures of gem-diamine and external aldimine complexes. Furthermore, the crystal structures reveal an unexpected allosteric product activator site for putrescine located on the 2-fold axis between the two active sites. Putrescine binds by donating hydrogen bonds from the ammonium groups to Asp-361 and Gln-358 in the specificity helix of both chains. Addition of 0.1–1 mM putrescine eliminates the lag in steady state kinetics and abolishes the sigmoid kinetics. The catalytic loop was modeled with AlphaFold2, and the model shows that Glu-181 can form additional hydrogen bonds with the bound putrescine, likely stabilizing the catalytic closed conformation.

Free Energy Landscape and Proton Transfer Pathways of the Transimination Reaction at the Active site of the Serine Hydroxymethyltransferase Enzyme in Aqueous Medium.

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme belonging to the fold type I or aspartate aminotransferase (AspAT) family of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. Like other PLP-dependent enzymes, SHMT also undergoes the so-called transimination reaction before exhibiting its enzymatic activity. The transimination process constitutes an important pre-step for all PLP-dependent enzymes, where an internal aldimine of the PLP-enzyme complex gets converted to an external aldimine of the substrate-PLP complex at the active site of the enzyme. In case of the transimination reaction involving SHMT, the PLP molecule bound to the active site lysine residue of SHMT (internal aldimine) gets detached from the enzyme by a serine substrate to produce an external aldimine complex, where the PLP is now bound to the serine substrate. In the current study, the free energy surfaces and reaction pathways of different steps of the transimination reaction at the active site of SHMT are investigated by employing hybrid quantum mechanical/molecular mechanical (QM/MM) simulations combined with metadynamics methods of rare event sampling. It is found that the process of transimination involving serine and PLP at the active site of the SHMT enzyme takes place through different elementary steps such as the formation of the first geminal diamine intermediate (GDI1), transfer of a proton from the substrate serine to the phenolic oxygen of PLP, followed by another proton transfer from PLP to the amine nitrogen of lysine with the formation of the second geminal diamine intermediate (GDI2), and finally, detachment of the active site lysine residue from PLP to produce the external aldimine.

Structural Basis of the Stereochemistry of Inhibition of Tryptophan Synthase by Tryptophan and Derivatives.

We have examined the reaction of Salmonella enterica serovar typhimurium tryptophan (Trp) synthase α2β2 complex with l-Trp, d-Trp, oxindolyl-l-alanine (OIA), and dioxindolyl-l-alanine (DOA) in the presence of disodium (dl)-α-glycerol phosphate (GP), using stopped-flow spectrophotometry and X-ray crystallography. All structures contained the d-isomer of GP bound at the α-active site. (3S)-OIA reacts with the pyridoxal-5'-phosphate (PLP) of Trp synthase to form a mixture of external aldimine and quinonoid complexes. The α-carboxylate of OIA rotates about 90° to become planar with the PLP when the quinonoid complex is formed, resulting in a conformational change in the loop of residues 110-115. The COMM domain of the Trp synthase-OIA complex is found as a mixture of two conformations. The (3R)-diastereomer of DOA binds about 5-fold more tightly than (3S)-OIA and also forms a mixture of aldimine and quinonoid complexes. DOA forms an additional H-bond between the 3-OH of DOA and βLys-87. l-Trp does not form a covalent complex with the PLP of Trp synthase. However, d-Trp forms a mixture of two external aldimine complexes which differ in the orientation of the α-carboxylate. In one conformation, the α-carboxylate is in the plane of the PLP, while in the other conformation, the α-carboxylate is perpendicular to the PLP plane. These results confirm that the stereochemistry of the transient indolenine quinonoid intermediate in the mechanism of Trp synthase is (3S) and demonstrate the linkage between aldimine and quinonoid reaction intermediates in the β-active site and allosteric communications with the α-active site.

Inhibition of tyrosine phenol-lyase by tyrosine homologues.

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 K i 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 K i values in the range of 0.8-1.5mM. 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.

Structural and Mutational Studies on Substrate Specificity and Catalysis of Salmonella typhimurium D-Cysteine Desulfhydrase

Salmonella typhimurium DCyD (StDCyD) is a fold type II pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. It also efficiently degrades β-chloro-D-alanine (βCDA). D-Ser is a poor substrate while the enzyme is inactive with respect to L-Ser and 1-amino-1-carboxy cyclopropane (ACC). Here, we report the X-ray crystal structures of StDCyD and of crystals obtained in the presence of D-Cys, βCDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS) at resolutions ranging from 1.7 to 2.6 Å. The polypeptide fold of StDCyD consisting of a small domain (residues 48–161) and a large domain (residues 1–47 and 162–328) resembles other fold type II PLP dependent enzymes. The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0–6.0 Å away from the active site. ACC forms an external aldimine complex while D- and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggest formation of PMP by the hydrolysis of cycloserines. Mutational studies suggest that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cloned and expressed in Escherichia coli. The purified enzyme was shown to be a dimeric protein with a monomeric molecular mass of 49 kDa. PvSHMT has a maximum absorption peak at 422 nm with a molar absorption coefficient of 6370 M(-1) x cm(-1). The K(d) for binding of the enzyme and pyridoxal-5-phosphate was 0.14 +/- 0.01 microM. An alternative assay for measuring the tetrahydrofolate-dependent SHMT activity based on the coupled reaction with 5,10-methylenetetrahydrofolate reductase (EC 1.5.1.20) from E. coli was developed. PvSHMT uses a ternary-complex mechanism with a k(cat) value of 0.98 +/- 0.06 s(-1) and K(m) values of 0.18 +/- 0.03 and 0.14 +/- 0.02 mM for L-serine and tetrahydrofolate, respectively. The optimum pH of the SHMT reaction was 8.0 and an Arrhenius's plot showed a transition temperature of 19 degrees C. Besides L-serine, PvSHMT forms an external aldimine complex with D-serine, L-alanine, L-threonine and glycine. PvSHMT also catalyzes the tetrahydrofolate-independent retro-aldol cleavage of 3-hydroxy amino acids. Although L-serine is a physiological substrate for SHMT in the tetrahydrofolate-dependent reaction, PvSHMT can also use D-serine. In the absence of tetrahydrofolate at high pH, PvSHMT forms an enzyme-quinonoid complex with D-serine, but not with L-serine, whereas SHMT from rabbit liver was reported to form an enzyme-quinonoid complex with L-serine. The substrate specificity difference between PvSHMT and the mammalian enzyme indicates the dissimilarity between their active sites, which could be exploited for the development of specific inhibitors against PvSHMT.

Pressure and Temperature Jump Relaxation Kinetics of the Conformational Change in Salmonella typhimurium Tryptophan Synthase l-Serine Complex: Large Activation Compressibility and Heat Capacity Changes Demonstrate the Contribution of Solvation

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.

Acceleration of the Substrate Cα Deprotonation by an Analogue of the Second Substrate Palmitoyl-CoA in Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Calpha of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated alpha-deprotonation indicates that the alpha-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the alpha-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Calpha-H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the alpha-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His(159) with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.

Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase,

Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.

Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase: SPECTROSCOPIC AND KINETIC STUDIES

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the mycobacterium tuberculosis KAPA synthase with both L- AND D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 m(-1) s(-1)) is approximately 5 times slower than that with L-alanine (k = 399.4 m(-1) s(-1)). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed d-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k = 16.9 x 10(4) m(-1) s(-1)). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, d-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (Ki = 114.83 microm) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 microm), the next enzyme of the pathway.

Differential Effects of Temperature and Hydrostatic Pressure on the Formation of Quinonoid Intermediates from l-Trp and l-Met by H463F Mutant Escherichia coli Tryptophan Indole-lyase

Escherichia coli tryptophan indole-lyase (Trpase) is a bacterial pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the reversible beta-elimination of l-Trp to give indole and ammonium pyruvate. H463F mutant E. coli Trpase (H463F Trpase) has very low activity with l-Trp, but it has near wild-type activity with other in vitro substrates, such as S-ethyl-l-cysteine and S-(o-nitrophenyl)-l-cysteine [Phillips, R. S., Johnson, N., and Kamath, A. V. (2002) Formation in vitro of Hybrid Dimers of H463F and Y74F Mutant Escherichia coli Tryptophan Indole-lyase Rescues Activity with l-Tryptophan, Biochemistry 41, 4012-4019]. The interaction of H463F Trpase with l-Trp and l-Met, a competitive inhibitor, has been investigated by rapid-scanning stopped-flow, high-pressure, and pressure jump spectrophotometry. Both l-Trp and l-Met bind to H463F Trpase to form equilibrating mixtures of external aldimine and quinonoid intermediates, absorbing at approximately 420 and approximately 505 nm, respectively. The apparent rate constant for quinonoid intermediate formation exhibits a hyperbolic dependence on l-Trp and l-Met concentration. The rate constant for quinonoid intermediate formation from l-Trp is approximately 10-fold lower for H463F Trpase than for wild-type Trpase, but the rate constant for reaction of l-Met is similar for H463F Trpase and wild-type Trpase. The temperature dependence of the rate constants for quinonoid intermediate formation reveals that both l-Trp and l-Met have similar values of DeltaH(++), but l-Met has a more negative value of DeltaS(++). Hydrostatic pressure perturbs the spectra of the H463F l-Trp and l-Met complexes, by shifting the position of the equilibria between different quinonoid and external aldimine complexes. Pressure-jump experiments show relaxations at 500 nm after rapid pressure changes of 100-400 bar with both l-Trp and l-Met. The apparent rate constants for relaxation of l-Trp, but not l-Met, show a significant increase with pressure. From these data, the value of DeltaV(++) for quinonoid intermediate formation from the external aldimine of l-Trp can be estimated to be -26.5 mL/mol, a larger than expected negative value for a proton transfer. These results suggest that there may be a contribution to the deprotonation reaction either from quantum mechanical tunneling or from a mechanical coupling of protein motion and proton transfer associated with the reaction of l-Trp, but not with l-Met.

Binding of C5-Dicarboxylic Substrate to Aspartate Aminotransferase: Implications for the Conformational Change at the Transaldimination Step,

The mechanism for the reaction of aspartate aminotransferase with the C4 substrate, l-aspartate, has been well established. The binding of the C4 substrate induces conformational change in the enzyme from the open to the closed form, and the entire reaction proceeds in the closed form of the enzyme. On the contrary, little is known about the reaction with the C5 substrate, l-glutamate. In this study, we analyzed the pH-dependent binding of 2-methyl-l-glutamate to the enzyme and showed that the interaction between the amino group of 2-methyl-l-glutamate and the pyridoxal 5'-phosphate aldimine is weak compared to that between 2-methyl-l-aspartate and the aldimine. The structures of the Michaelis complexes of the enzyme with l-aspartate and l-glutamate were modeled on the basis of the maleate and glutarate complex structures of the enzyme. The result showed that l-glutamate binds to the open form of the enzyme in an extended conformation, and its alpha-amino group points in the opposite direction of the aldimine, while that of l-aspartate is close to the aldimine. These models explain the observations for 2-methyl-l-glutamate and 2-methyl-l-aspartate. The crystal structures of the complexes of aspartate aminotransferase with phosphopyridoxyl derivatives of l-glutamate, d-glutamate, and 2-methyl-l-glutamate were solved as the models for the external aldimine and ketimine complexes of l-glutamate. All the structures were in the closed form, and the two carboxylate groups and the arginine residues binding them are superimposable on the external aldimine complex with 2-methyl-l-aspartate. Taking these facts altogether, it was strongly suggested that the binding of l-glutamate to aspartate aminotransferase to form the Michaelis complex does not induce a conformational change in the enzyme, and that the conformational change to the closed form occurs during the transaldimination step. The hydrophobic residues of the entrance of the active site, including Tyr70, are considered to be important for promoting the transaldimination process and hence the recognition of the C5 substrate.

Role of Lys-226 in the Catalytic Mechanism of Bacillus Stearothermophilus Serine HydroxymethyltransferaseCrystal Structure and Kinetic Studies,

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the reversible conversion of l-Ser and tetrahydropteroylglutamate (H(4)PteGlu) to Gly and 5,10-methylene tetrahydropteroylglutamate (CH(2)-H(4)PteGlu). Biochemical and structural studies on this enzyme have implicated several residues in the catalytic mechanism, one of them being the active site lysine, which anchors PLP. It has been proposed that this residue is crucial for product expulsion. However, in other PLP-dependent enzymes, the corresponding residue has been implicated in the proton abstraction step of catalysis. In the present investigation, Lys-226 of Bacillus stearothermophilus SHMT (bsSHMT) was mutated to Met and Gln to evaluate the role of this residue in catalysis. The mutant enzymes contained 1 mol of PLP per mol of subunit suggesting that Schiff base formation with lysine is not essential for PLP binding. The 3D structure of the mutant enzymes revealed that PLP was bound at the active site in an orientation different from that of the wild-type enzyme. In the presence of substrate, the PLP ring was in an orientation superimposable with that of the external aldimine complex of wild-type enzyme. However, the mutant enzymes were inactive, and the kinetic analysis of the different steps of catalysis revealed that there was a drastic reduction in the rate of formation of the quinonoid intermediate. Analysis of these results along with the crystal structures suggested that K-226 is responsible for flipping of PLP from one orientation to another which is crucial for H(4)PteGlu-dependent Calpha-Cbeta bond cleavage of l-Ser.

Spectrophotometric and Steady-State Kinetic Analysis of the Biosynthetic Arginine Decarboxylase of Yersinia pestis Utilizing Arginine Analogues as Inhibitors and Alternative Substrates

The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, l-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate.

Structural studies of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: the apo, substrate, and product-aldimine complexes.

The evolution of biosynthetic pathways is difficult to reconstruct in hindsight; however, the structures of the enzymes that are involved may provide insight into their development. One enzyme in the cobalamin biosynthetic pathway that appears to have evolved from a protein with different function is L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica, which is structurally similar to histidinol phosphate aminotransferase [Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C., and Rayment, I. (2002) Biochemistry 41, 4798-4808]. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. To understand the relationship between this decarboxylase and the aspartate aminotransferase family to which it belongs, the structures of CobD in its apo state, the apo state complexed with the substrate, and its product external aldimine complex have been determined at 1.46, 1.8, and 1.8 A resolution, respectively. These structures show that the enzyme steers the breakdown of the external aldimine toward decarboxylation instead of amino transfer by positioning the carboxylate moiety of the substrate out of the plane of the pyridoxal ring and by placing the alpha-hydrogen out of reach of the catalytic base provided by the lysine that forms the internal aldimine. It would appear that CobD evolved from a primordial PLP-dependent aminotransferase, where the selection was based on similarities between the stereochemical properties of the substrates rather than preservation of the fate of the external aldimine. These structures provide a sequence signature for distinguishing between L-threonine-O-3-phosphate decarboxylase and histidinol phosphate aminotransferases, many of which appear to have been misannotated.

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies.

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

Acid-base chemistry of the reaction of aromatic L-amino acid decarboxylase and dopa analyzed by transient and steady-state kinetics: preferential binding of the substrate with its amino group unprotonated.

Transient and steady-state kinetic analysis of the reaction of aromatic L-amino acid decarboxylase (AADC), a pyridoxal 5'-phosphate- (PLP-) dependent enzyme, with its substrate dopa was carried out at various pH. The association of AADC and dopa to form the Michaelis complex and the subsequent transaldimination reaction to form the dopa-PLP Schiff base (external aldimine) were followed with a stopped-flow spectrophotometer. Combined with the steady-state k(cat) value, we could present a minimum mechanism for the reaction of AADC and dopa. In the mechanism, the association of the aldimine-protonated form of the enzyme (EH(+)) and the alpha-amino-group-unprotonated form of the substrate (S) is the main route leading to the Michaelis complex. In addition, the association of EH(+) and the alpha-amino-group-protonated form of the substrate (SH(+)) to form a Michaelis complex EH(+).SH(+) was also found as a minor route. The pK(a) of the alpha-amino group of dopa was expected to be decreased in the Michaelis complex, promoting the conversion of EH(+).SH(+) to EH(+).S, the species that directly undergoes transaldimination to form the external aldimine complex. The association of EH(+) and S had been identified as a minor route for the reaction of aspartate and aspartate aminotransferase (AspAT), which has an unusually low pK(a) value of the aldimine and can use the aldimine-unprotonated form (E) of the enzyme for adsorbing the prevalent species SH(+) [Hayashi and Kagamiyama (1997) Biochemistry 36, 13558-13569]. The present study implies that, in most PLP enzymes that have a high pK(a) value of the aldimine like AADC, S preferentially binds to the enzyme (EH(+)). The minor route of EH(+) + SH(+) in AADC may be related to the flexibility of the protein in the Michaelis complex, and a simulation analysis showed that the presence of this route decreases the k(cat) value while increasing the k(cat)/K(m) value. It also suggested that AADC has evolved to suppress the minor route to the extent necessary to obtain the maximal k(cat) value at neutral pH.

Binding of phenol and analogues to alanine complexes of tyrosine phenol-lyase from Citrobacter freundii: implications for the mechanisms of alpha,beta-elimination and alanine racemization.

We have examined the interaction of Citrobacter freundii tyrosine phenol-lyase with both L- and D-alanine. This enzyme catalyzes the racemization of alanine as a side reaction, in addition to the physiological beta-elimination of L-tyrosine to give phenol and ammonium pyruvate. The steady-state kinetic parameters for alanine racemization, kcat and Km, for D-alanine are 0.008 S-1 and 32 mM, respectively, while those for L-alanine are 0.03 S-1 and 11 mM. Incubation of tyrosine phenol-lyase with either L- or D-alanine forms a quinonoid complex that exhibits a strong peak at 500 nm. The presence of K+ increases the intensity of the 500-nm absorption with L-alanine, but decreases the intensity of the peak with D-alanine. Rate constants for the formation of these quinonoid intermediates and the effects of phenol and analogues on the reaction with either L- or D-alanine have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Phenol binds to all the intermediates of tyrosine phenol-lyase with L- and D-alanine, but most strongly to the external aldimine complex, resulting in a decrease in the absorbance at 500 nm at equilibrium. Pyridine N-oxide binds selectively to the quinonoid complex of alanine, and thus causes an increase in the absorbance at 500 nm at equilibrium. 4-Hydroxypyridine causes a decrease in absorbance at 500 nm during the fast phase, but an increase in absorbance at 502 nm in a subsequent slow relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli serine hydroxymethyltransferase. The role of histidine 228 in determining reaction specificity.

Serine hydroxymethyltransferase has a conserved histidine residue (His-228) next to the lysine residue (Lys-229) which forms the internal aldimine with pyridoxal 5'-phosphate. This histidine residue is also conserved at the equivalent position in all amino acid decarboxylases and tryptophan synthase. Two mutant forms of Escherichia coli serine hydroxymethyltransferase, H228N and H228D, were constructed, expressed, and purified. The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady state kinetics, and rapid reaction kinetics. The conclusions of these studies were that His-228 plays an important role in the binding and reactivity of the hydroxymethyl group of serine in the one-carbon-binding site. The mutant enzymes utilize substrates and substrate analogs more effectively for a variety of alternate non-physiological reactions compared to the wild type enzyme. As one example, the mutant enzymes cleave L-serine to glycine and formaldehyde when tetrahydropyteroylglutamate is replaced by 5-formyltetrahydropteroylglutamate. The released formaldehyde inactivates these mutant enzymes. The loss of integrity of the one-carbon-binding site with L-serine in the two mutant forms of the enzyme may be the result of these enzymes not undergoing a conformational change to a closed form of the active site when serine forms the external aldimine complex.

Reaction of indole and analogs with amino acid complexes of Escherichia coli tryptophan indole-lyase: detection of a new reaction intermediate by rapid-scanning stopped-flow spectrophotometry

The effects of indole and analogues on the reaction of Escherichia coli tryptophan indole-lyase (tryptophanase) with amino acid substrates and quasisubstrates have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Indole binds rapidly (within the dead time of the stopped-flow instrument) to both the external aldimine and quinonoid complexes with L-alanine, and the absorbance of the quinonoid intermediate decreases in a subsequent slow relaxation. Indoline binds preferentially to the external aldimine complex with L-alanine, while benzimidazole binds selectively to the quinonoid complex of L-alanine. Indole and indoline do not significantly affect the spectrum of the quinonoid intermediates formed in the reaction of the enzyme with S-alkyl-L-cysteines, but benzimidazole causes a rapid decrease in the quinonoid peak at 512 nm and the appearance of a new peak at 345 nm. Benzimidazole also causes a rapid decrease in the quinonoid peak at 505 nm formed in the reaction with L-tryptophan and the appearance of a new absorbance peak at 345 nm. Furthermore, addition of benzimidazole to solutions of enzyme, potassium pyruvate, and ammonium chloride results in the formation of a similar absorption peak at 340 nm. This complex reacts rapidly with indole to form a quinonoid intermediate very similar to that formed from L-tryptophan. This new intermediate is formed faster than catalytic turnover (kcat = 6.8 s-1) and may be an alpha-aminoacrylate intermediate bound as a gem-diamine.