title: A mutant D-amino acid aminotransferase with broad substrate specificity: construction by replacement of the interdomain loop Pro119-Arg120-Pro121 by Gly-Gly-Gly.
abstract: 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.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Gutierrez
authors.forename: T
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Y
authors.initials: Y
authors.lastname: Fuchikami
authors.forename: K
authors.initials: K
authors.lastname: Soda
authors.forename: N
authors.initials: N
authors.lastname: Esaki
mesh_terms: D019943:Amino Acid Substitution; D019881:Aminoacyltransferases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D002942:Circular Dichroism; D007700:Kinetics; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D013438:Sulfhydryl Compounds; D019881:Aminoacyltransferases; C103932:D-amino acid transferase, Saccharothrix