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PDBsum entry 1szu
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References listed in PDB file
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Key reference
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Title
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Kinetic and crystallographic analysis of active site mutants of escherichia coli gamma-Aminobutyrate aminotransferase.
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Authors
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W.Liu,
P.E.Peterson,
J.A.Langston,
X.Jin,
X.Zhou,
A.J.Fisher,
M.D.Toney.
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Ref.
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Biochemistry, 2005,
44,
2982-2992.
[DOI no: ]
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PubMed id
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Abstract
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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.
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