Catalytic ability and stability of two recombinant mutants of D‐amino acid transaminase involved in coenzyme binding

Abstract

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, Le., 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, 0-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.

Site‐directed mutagenesis of the amino acid residues in β‐strand III [Val30‐Val36] of d‐amino acid aminotransferase of Bacillus sp. YM‐1

“…The phenolic oxygen of Tyr 3a residue was postulated to form hydrogen bond with the 3'-oxygen of PLP [5]. Thus, substitution of this residue with alanine resulted in a marked decrease in kcat, showing results similar to those for the Y31Q mutant [6]. Increased Km for ct-ketoglutarate may be attributed to the enhanced mobility of the cofactor in the active site as in the Y31Q mutant.…”

“…The functional role of the Tyr 31 residue has been studied by other researchers [6]. Therefore, we tried to investigate the characteristics of Glu32-substituted enzymes as an approach to demonstration of the role of the Glu 32 residue.…”

“…The Tyr 31 residue is known to interact with the 3'-oxygen of pyridoxal 5'-phosphate (PLP), a cofactor required by the mutant [5,6]. These results led us to speculate that these amino acid residues might play an important role in enzyme catalysis.…”

“…We have been focusing on random mutagenesis of the enzyme to improve the catalytic properties and observed that substitution of alanine for the Va133 residue resulted in a significant change in the catalytic properties of the enzyme (unpublished result). The Tyr 31 residue is known to interact with the 3'-oxygen of pyridoxal 5'-phosphate (PLP), a cofactor required by the mutant [5,6]. These results led us to speculate that these amino acid residues might play an important role in enzyme catalysis.…”

Overexpression of a newly identified d‐amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion

“…The ADCL protein in E. coli and MSMEG_5795 share 29% identity, higher than the 23% identity shared by the d‐amino acid transaminase of B. subtilis and MSMEG_5795, which likely explains the predominant annotation of MSMEG_5795 as an ADCL. Notably, the d‐AAT activity we measured for the MSMEG_5795 protein is significant at V max of 13.8 U mg −1 but is less than the V max of 200 U mg −1 reported for the Bacillus enzyme (Van Ophem et al ., ). This could explain why wild‐type MSMEG_5795 under control of its native promoter is unable to rescue Δ murI or Δ alr deletion strains.…”