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Journal of Biochemistry Advance Access published online on March 27, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm082
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© 2007 The Japanese Biochemical Society

The effects of mutations at position 253 on the thermostability of the Bacillus subtilis 3-isopropylmalate dehydrogenase subunit interface.

Takatoshi Ohkuri1,2 and Akihiko Yamagishi1,*

1Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
2Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan

* To whom all correspondence should be addressed. Akihiko Yamagishi, JDepartment of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan, Phone: 81-426-76-7139, Fax: 84-426-76-7145, E-mail: yamagish{at}ls.toyaku.ac.jp

Received December 13, 2006; Accepted March 14, 2007


  Abstract

3-isopropylmalate dehydrogenase (IPMDH) is a dimeric enzyme with a strongly hydrophobic core that is composed of residues from four {alpha}-helices. We replaced Glu253, which is found in the hydrophobic core and is part of the subunit interface of the Bacillus subtilis (Bs) IPMDH, with several other amino acids to probe. The thermostabilities of the mutants were assessed by measuring the residual enzymatic activities at 40 °C after heat treatment and by monitoring changes in ellipticity at 222 nm as the environmental temperature increased incrementally. The results of these studies indicate that, for residues with non-polar side chains, when positioned at residue 253, the thermostabilities of their corresponding mutants correlate positively with the relative hydrophobicities of the side chains. Relative activities of all mutants are lower than that of the wild type enzyme. For two of the mutants, we directly show that the substitution at position 253 negatively affects Mn2+ binding, which is required for catalysis. When a lysine is the position 253 residue, the protein dissociates. The results presented herein increase our understanding of the role played by the BsIPMDH dimer interface on the stability and activity of BsIPMDH.

Key Words: isopropylmalate dehydrogenase, thermostability, subunit interaction, hydrophobic core, dimeric enzyme


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