title: Kinetic and crystallographic analysis of active site mutants of Escherichia coli gamma-aminobutyrate aminotransferase.
abstract: The E. coli isozyme of gamma-aminobutyrate aminotransferase (GABA-AT) is a tetrameric pyridoxal phosphate-dependent enzyme that catalyzes transamination between primary amines and alpha-keto acids. The roles of the active site residues V241, E211, and I50 in the GABA-AT mechanism have been probed by site-directed mutagenesis. The beta-branched side chain of V241 facilitates formation of external aldimine intermediates with primary amine substrates, while E211 provides charge compensation of R398 selectively in the primary amine half-reaction and I50 forms a hydrophobic lid at the top of the substrate binding site. The structures of the I50Q, V241A, and E211S mutants were solved by X-ray crystallography to resolutions of 2.1, 2.5, and 2.52 A, respectively. The structure of GABA-AT is similar in overall fold and active site structure to that of dialkylglycine decarboxylase, which catalyzes both transamination and decarboxylation half-reactions in its normal catalytic cycle. Therefore, an attempt was made to convert GABA-AT into a decarboxylation-dependent aminotransferase similar to dialkylglycine decarboxylase by systematic mutation of E. coli GABA-AT active site residues. Two of the twelve mutants presented, E211S/I50G/C77K and E211S/I50H/V80D, have approximately 10-fold higher decarboxylation activities than the wild-type enzyme, and the E211S/I50H/V80D has formally changed the reaction specificity to that of a decarboxylase.
authors.affiliation: Department of Chemistry, University of California-Davis, Davis, California 95616, USA.
authors.forename: Wenshe
authors.initials: W
authors.lastname: Liu
authors.forename: Peter E
authors.initials: PE
authors.lastname: Peterson
authors.forename: James A
authors.initials: JA
authors.lastname: Langston
authors.forename: Xueguang
authors.initials: X
authors.lastname: Jin
authors.forename: Xianzhi
authors.initials: X
authors.lastname: Zhou
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Fisher
authors.forename: Michael D
authors.initials: MD
authors.lastname: Toney
mesh_terms: D000612:4-Aminobutyrate Transaminase / Q000737:chemistry* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001667:Binding, Competitive; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D000612:4-Aminobutyrate Transaminase