Different modes of action of inhibitors of bacterial D-amino acid transaminase. A target enzyme for the design of new antibacterial agents.
(17/31)
D-Amino acid transaminase from Bacillus sphaericus shows a deuterium kinetic isotope effect (VH/VD) between 2 and 3 in the transamination of alpha-protio- or alpha-deuterio-D-alanine and alpha-ketoglutarate, suggesting that alpha-proton abstraction is at least partially rate-limiting for this reaction. This transaminase also catalyzes a beta-elimination reaction with substrates such as beta-fluoroalanine with no detectable deuterium isotope effect (VH/BD = 1). These results, taken together with previous work (Soper, T. S., and Manning, J. M. (1978) Biochemistry 17, 3377-3384) suggest that the rate-limiting step in the beta-elimination reaction is solvolysis of an alpha-aminoacrylate-pyridoxal-P Schiff's base intermediate. D-Cycloserine is an active site titrant of D-amino acid transaminase. Inactivation by cycloserine can be completely reversed by dialysis against pyridoxal phosphate at neutral pH. Gabaculine is also an efficient inhibitor of this enzyme and possesses some antibacterial activity. The latter two inhibitors probably act by sequestration of the coenzyme rather than by alkylation of the protein as with the beta-halo derivatives of D-alanine. (+info)
Role of leucine 201 of thermostable D-amino acid aminotransferase from a thermophile, Bacillus sp. YM-1.
(18/31)
We studied the catalytic role of leucine 201 residue of the thermostable D-amino acid aminotransferase: the residue was shown crystallographically to be in the vicinity of the active site to interact with the bound pyridoxal phosphate. We replaced the leucine 201 by alanyl or tryptophanyl residues by means of site-directed mutagenesis. The L201A and L201W mutant enzymes showed anomalous kinetic behavior in the overall reaction. The reaction rates of the L201A and L201W mutant enzymes gradually decreased with an increase in the reaction time to become practically zero at a high concentration of substrates. The mutant enzymes were also inactivated in the half reaction with D-alanine, although more slowly than in the overall reaction. The absorption spectra of the mutant enzymes in the presence of D-alanine and alpha-ketoglutarate suggest that the enzyme molecules were mostly in the pyridoxamine form under the conditions employed. These phenomena were explained by assuming two (or more) enzyme species showing kinetically different catalysis for pyridoxamine form of the mutant enzymes, and the rate of conversion from one of these pyridoxamine forms to the pyridoxal form should be very low. The leucine 201 residue probably regulates the function of cofactor during the reaction of D-amino acid aminotransferase. (+info)
Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase.
(19/31)
Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene). (+info)
Role reversal for substrates and inhibitors. Slow inactivation of D-amino acid transaminase by its normal substrates and protection by inhibitors.
(20/31)
D-Amino acid transaminase, which catalyzes the synthesis of D-alanine and D-glutamate for the bacterial cell wall, is a candidate for the design of specific inhibitors that could be novel antimicrobial agents. Under the experimental conditions usually employed for enzyme assays, kinetic parameters for its substrates were determined for short incubation periods, when intermediates and products do not accumulate and the enzyme activity is linear with time. Such kinetic analyses indicate that the enzyme accepts most D-amino acids but D-aspartate and D-glutamate are the best substrates. Under a different type of experimental conditions when the enzyme is exposed to D-alanine, intermediates, and products for periods of hours, it slowly becomes inactivated (Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., and Manning, J. M. (1992) Biochemistry 31, 6018-6023). We now report that D-aspartate, D-glutamate, and L-alanine also lead to slow inactivation. Methylation or amidation of the alpha-COOH group of D-alanine prevents inactivation, indicating that decarboxylation is required for inactivation; the slow release of CO2 from substrate is demonstrated. The alpha-methyl analog of D-alanine, D-aspartate, and D-glutamate do not lead to inactivation, showing that the alpha-hydrogen of the substrate is required, i.e. that some processing is required. Lys145, which binds pyridoxal 5'-phosphate in the wild-type enzyme, is not involved in the inactivation since two active site mutant enzymes, K145Q and K145N, are also inactivated. Reactivation of the inactive enzyme at acidic pH is accompanied by the release of ammonia corresponding to 1 mol/mol of dimeric enzyme. Competitive inhibitors, amine-containing buffers, and thiols effectively impede the inactivation. This reversal in the roles of substrates and inhibitors, i.e. when a substrate can be an inactivator and an inhibitor can act as a protector, occurs during a time period not usually used to measure steady-state kinetics or initial velocities of enzyme reactions and could have physiological relevance in cells. (+info)
Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase.
(21/31)
D-Amino acid transaminase (EC 2.6.1.21), from Bacillus sp. YM-1, a thermostable enzyme with pyridoxal 5'-phosphate as coenzyme and a target for the design of novel antimicrobial agents, catalyzes the reversible transfer of an amino group between D-alanine and alpha-ketoglutarate to form pyruvate and D-glutamate, respectively. To explore the catalytic role of Lys-145, which binds the coenzyme, a site-specific mutant enzyme, K145Q (in which Lys-145 had been mutated to glutamine) constructed earlier (Futaki, S., Ueno, H., Martinez del Pozo, A., Pospischil, M. A., Manning, J. M., Ringe, D., Stoddard, B., Tanizawa, K., Yoshimura, T., and Soda, K. (1990) J. Biol. Chem. 265, 22306-22312) was compared to the wild-type enzyme for its kinetic parameters. Initial velocity studies and partial reaction isotope exchange experiments showed that the low activity of the mutant enzyme (about 1.5% the activity of the wild-type enzyme with saturating substrates) is an intrinsic property, confirming that contaminating enzymes do not account for the low activity of the K145Q mutant enzyme. The rates of the forward reaction for both wild-type and mutant enzymes were 30-40 times higher than the rates of the reverse reaction. KM values for the four substrates were 10 to 100 higher for the mutant compared to the wild-type enzyme. Whereas D-alanine is preferred over L-alanine by the wild-type enzyme (10(3) higher kcat/KM for D- over L-alanine), the K145Q enzyme does not efficiently discriminate between L- and D-alanine. Both wild-type and mutant enzymes also catalyze the slow racemization of L- and D-alanine. Proton NMR studies showed that wild-type enzyme catalyzed a time-dependent exchange of the C alpha proton of D-alanine with solvent D2O and a slow exchange of the alpha proton of L-alanine; the latter slow exchange rate is the same for the C alpha proton of both L- and D-alanine with the K145Q mutant enzyme. Thus, in addition to binding pyridoxal 5'-phosphate, the active-site Lys-145 of D-amino acid transaminase is involved in several other important functions, i.e. it optimizes catalytic efficiency and it maintains stereochemical fidelity. The steady-state kinetic results on the K145Q mutant enzyme together with the findings on the relative racemization rates and the NMR protein exchange data suggest that an alternate base catalyzes abstraction of the alpha proton of substrate in this mutant D-amino acid transaminase. (+info)
Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.
(22/31)
Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, beta-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme. (+info)
Pyridoxal 5'-phosphate binding of a recombinant rat serine: pyruvate/alanine:glyoxylate aminotransferase.
(23/31)
Serine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV amino-transferase. The present study was undertaken to characterize the pyridoxal 5'-phosphate (PLP) binding to a recombinant rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits. Purified SPT10 exhibited absorption maxima at approximately 330 nm in addition to a 278 nm protein peak and a approximately 420 nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit. Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at approximately 420 nm, but still showed the approximately 330 nm peak. Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of approximately 0.1 microM, resulting in a holo-SPT10 preparation whose specific activity and A approximately 420/A approximately 330 absorbance ratio were higher than those of the original SPT10. On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10, 22-23 kDa fragments migrated as a pair of bands. On amino acid sequencing, the approximately 22 and approximately 23 kDa pair gave the same sequence, except that Lys was released only from the approximately 22 kDa band material in the cycle corresponding to Lys209. NaB3H4-treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the approximately 45 kDa band. It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330 nm-absorbing chromophore. (+info)
Site-directed mutagenesis of the amino acid residues in beta-strand III [Val30-Val36] of D-amino acid aminotransferase of Bacillus sp. YM-1.
(24/31)
The beta-strand III formed by amino acid residues Val30-Val36 is located across the active site of the thermostable D-amino acid aminotransferase (D-AAT) from thermophilic Bacillus sp. YM-1, and the odd-numbered amino acids (Tyr31, Val33, Lys35) in the strand are revealed to be directed toward the active site. Interestingly, Glu32 is also directed toward the active site. We first investigated the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a marked decrease in k(cat) value, retaining less than 1% of the wild-type enzyme activity. The k(cat) values of V33A and K35A were changed slightly, but the Km of K35A for alpha-ketoglutarate was increased to 35.6 mM, compared to the Km value of 2.5 mM for the wild-type enzyme. These results suggested that the positive charge at Lys35 interacted electrostatically with the negative charge at the side chain of alpha-ketoglutarate. Site-directed mutagenesis of the Glu32 residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu32-substituted enzymes, the Glu32 residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5'-phosphate (PLP) and the epsilon-amino group of the Lys145 residue. (+info)