1TOK

Maleic acid-bound structure of SRHEPT mutant of E. coli aspartate aminotransferase

Summary for 1TOK

• Entry DOI: 10.2210/pdb1tok/pdb
• Related: 1TOE 1TOG 1TOI 1TOJ
• Descriptor: Aspartate aminotransferase, MALEIC ACID (3 entities in total)
• Functional Keywords: aspartate aminotransferase hexamutant, srhept, transferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P00509
• Total number of polymer chains: 2
• Total formula weight: 87882.71

Authors

Chow, M.A.,McElroy, K.E.,Corbett, K.D.,Berger, J.M.,Kirsch, J.F. (deposition date: 2004-06-14, release date: 2004-10-05, Last modification date: 2023-11-15)

Primary citation

Chow, M.A.,McElroy, K.E.,Corbett, K.D.,Berger, J.M.,Kirsch, J.F.
Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase
Biochemistry, 43:12780-12787, 2004

PubMed 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 k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) 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.

PubMed: 15461450
DOI: 10.1021/bi0487544

Experimental method

X-RAY DIFFRACTION (1.85 Å)