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

• Published in: Protein engineering 1998-01, Vol.11 (1), p.53-58
• Main Authors: Gutierrez, A, Yoshimura, T, Fuchikami, Y, Soda, K, Esaki, N
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Aminoacyltransferases - chemistry; Aminoacyltransferases - metabolism; Bacterial Proteins; Circular Dichroism; Kinetics; Protein Conformation; Spectrophotometry, Ultraviolet; Substrate Specificity; Sulfhydryl Compounds - chemistry; Temperature; Thermodynamics
• ISSN: 0269-2139; 1741-0126; 1741-0134
• DOI: 10.1093/protein/11.1.53

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