Narrowing substrate specificity in a directly evolved enzyme: The A293D mutant of aspartate aminotransferase

Margaret A. Chow, Kathryn E. McElroy, Kevin D. Corbett, James M Berger, Jack F. Kirsch

Research output: Contribution to journalArticle

Abstract

Several mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of kcat/KmPhe to kcat/KmAsp as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release.

Original languageEnglish (US)
Pages (from-to)12780-12787
Number of pages8
JournalBiochemistry®
Volume43
Issue number40
DOIs
StatePublished - Oct 12 2004
Externally publishedYes

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Aspartate Aminotransferases
Substrate Specificity
Escherichia coli
Tyrosine Transaminase
Conformations
Substitution reactions
Substrates
Enzymes
Mutagenesis
Mutation
Site-Directed Mutagenesis
Phenylalanine
Aspartic Acid
Scaffolds
Catalytic Domain
Crystal structure
Ligands
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Narrowing substrate specificity in a directly evolved enzyme : The A293D mutant of aspartate aminotransferase. / Chow, Margaret A.; McElroy, Kathryn E.; Corbett, Kevin D.; Berger, James M; Kirsch, Jack F.

In: Biochemistry®, Vol. 43, No. 40, 12.10.2004, p. 12780-12787.

Research output: Contribution to journalArticle

Chow, Margaret A. ; McElroy, Kathryn E. ; Corbett, Kevin D. ; Berger, James M ; Kirsch, Jack F. / Narrowing substrate specificity in a directly evolved enzyme : The A293D mutant of aspartate aminotransferase. In: Biochemistry®. 2004 ; Vol. 43, No. 40. pp. 12780-12787.
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