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

Wenshe Liu, Peter E. Peterson, James A. Langston, Xueguang Jin, Xianzhi Zhou, Andrew J Fisher, Michael D. Toney

Research output: Contribution to journalArticle

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Abstract

The E. coli isozyme of γ-aminobutyrate aminotransferase (GABA-AT) is a tetrameric pyridoxal phosphate-dependent enzyme that catalyzes transamination between primary amines and α-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 β-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 150 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 Å, 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 ∼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.

Original languageEnglish (US)
Pages (from-to)2982-2992
Number of pages11
JournalBiochemistry
Volume44
Issue number8
DOIs
StatePublished - Mar 1 2005

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4-Aminobutyrate Transaminase
2,2-dialkylglycine decarboxylase
gamma-Aminobutyric Acid
Escherichia coli
Catalytic Domain
Decarboxylation
Kinetics
Amines
Keto Acids
Mutagenesis
Pyridoxal Phosphate
Carboxy-Lyases
X ray crystallography
X Ray Crystallography
Substrates
Enzymes
Site-Directed Mutagenesis
Transaminases
Isoenzymes
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and crystallographic analysis of active site mutants of Escherichia coli γ-aminobutyrate aminotransferase. / Liu, Wenshe; Peterson, Peter E.; Langston, James A.; Jin, Xueguang; Zhou, Xianzhi; Fisher, Andrew J; Toney, Michael D.

In: Biochemistry, Vol. 44, No. 8, 01.03.2005, p. 2982-2992.

Research output: Contribution to journalArticle

Liu, Wenshe ; Peterson, Peter E. ; Langston, James A. ; Jin, Xueguang ; Zhou, Xianzhi ; Fisher, Andrew J ; Toney, Michael D. / Kinetic and crystallographic analysis of active site mutants of Escherichia coli γ-aminobutyrate aminotransferase. In: Biochemistry. 2005 ; Vol. 44, No. 8. pp. 2982-2992.
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AB - The E. coli isozyme of γ-aminobutyrate aminotransferase (GABA-AT) is a tetrameric pyridoxal phosphate-dependent enzyme that catalyzes transamination between primary amines and α-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 β-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 150 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 Å, 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 ∼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.

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