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Journal of Biochemistry Advance Access published online on April 19, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn053
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© 2008 The Japanese Biochemical Society

15N NMR Relaxation Studies of Y14F Mutant of Ketosteroid Isomerase: The Influence of Mutation on Backbone Mobility

Hyeong Ju Lee1, Ye Jeong Yoon1, Do Soo Jang2,{dagger}, Chul Kim1,{ddagger}, Hyung Jin Cha2, Bee Hak Hong2, Kwan Yong Choi2,* and Hee Cheon Lee1,*

1Department of Chemistry, 2Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, 790-784 Republic of Korea

*Author to whom correspondence should be addressed: Prof. Hee Cheon Lee Department of Chemistry, Pohang University of Science and Technology, Pohang, 790-784 Republic of Korea. E-mail: hcl{at}postech.ac.kr, Fax: 82-54-279-3399. Kwan Yong Choi, Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, 790-784 Republic of Korea, E-mail: kchoi{at}postech.ac.kr, Fax: 82-54-279-2199

Received March 4, 2008; Accepted April 8, 2008


  Abstract

Summary

The backbone dynamics of Y14F mutant of {triangleup}5-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38, and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.

Key Words: Backbone dynamics, Ketosteroid isomerase, Mutant, NMR, Relaxation

{dagger} Present address: The J. David Gladstone Institute, SF, CA94158, USA

{ddagger}Present address: 012 Fairchild Library, Div. of Chemistry and Chemical Engineering,


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