Kinetic and crystallographic analysis of active site mutants of Escherichia coli gamma-aminobutyrate aminotransferase.

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Liu W, Peterson PE, Langston JA, Jin X, Zhou X, Fisher AJ, Toney MD

Kinetic and crystallographic analysis of active site mutants of Escherichia coli gamma-aminobutyrate aminotransferase.

Biochemistry. 2005 Mar 1;44(8):2982-92.

PubMed ID

15723541 [ View in PubMed

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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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Pyridoxal phosphate	4-aminobutyrate aminotransferase, mitochondrial	Protein	Humans	Unknown	Inhibitor	Details

Polypeptides

Name	UniProt ID
4-aminobutyrate aminotransferase GabT	P22256	Details