1YOO

ASPARTATE AMINOTRANSFERASE MUTANT ATB17 WITH ISOVALERIC ACID

Summary for 1YOO

• Entry DOI: 10.2210/pdb1yoo/pdb
• Descriptor: ASPARTATE AMINOTRANSFERASE, PYRIDOXAL-5'-PHOSPHATE, ISOVALERIC ACID, ... (4 entities in total)
• Functional Keywords: aminotransferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P00509
• Total number of polymer chains: 1
• Total formula weight: 43968.46

Authors

Oue, S.,Okamoto, A.,Yano, T.,Kagamiyama, H. (deposition date: 1998-06-26, release date: 1999-02-02, Last modification date: 2023-08-09)

Primary citation

Oue, S.,Okamoto, A.,Yano, T.,Kagamiyama, H.
Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues.
J.Biol.Chem., 274:2344-2349, 1999

PubMed Abstract: Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-A resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.

PubMed: 9891001
DOI: 10.1074/jbc.274.4.2344

Experimental method

X-RAY DIFFRACTION (2.4 Å)