A mutant D-amino acid aminotransferase with broad substrate specificity: construction by replacement of the interdomain loop Pro119- Arg120-Pro121 by Gly-Gly-Gly

D-amino acid aminotransferase (EC 2.6.1.21) catalyzes the interconversionof various D-amino acids and 2-oxo acids. Each homodimer subunit consistsof two domains, which are connected by a single loop,Asn118-Pro119-Arg120-Pro121. The loop has no direct contact with the activesite region or the cofactor, pyridoxal 5'- phosphate. We attempted toincrease the conformational flexibility of this loop through a tripleglycine substitution. The resultant mutant P119G-R120G-P121G has featuresclearly different from the wild-type enzyme under overall as well ashalf-reaction conditions. The pre- steady-state kinetic analyses of halfreactions showed that the mutant enzyme has kmax values higher than thewild-type enzyme towards most D- amino acids examined. A concomitantdecrease in substrate affinity (1/Kd), particularly for acidic amino acids,was also observed. A putative binding site for the distal carboxyl group ofacidic amino acids in the wild-type enzyme was incidentally displaced bythe loop mutation, indicating a functional linkage between the interdomainloop and the active site region. This study has exemplified the usefulnessof engineering relatively distant loops as a means to modify substratespecificity of an enzyme.