Journal of Biochemistry Advance Access originally published online on April 19, 2008
Journal of Biochemistry 2008 144(2):159-166; 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
1Department of Chemistry; and 2Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, 790-784 Republic of Korea
Correspondence may also be addressed. Tel: +82-54-279-2295, Fax: +82 54 279 2199, E-mail: kchoi{at}postech.ac.kr
To whom correspondence should be addressed. Tel: +82-54-279-2116, Fax: +82 54 279 3399, E-mail: hcl{at}postech.ac.kr
Received March 4, 2008; Accepted April 8, 2008
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Abstract |
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The backbone dynamics of Y14F mutant of 
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
Abbreviations: CPMG, Carr–Purcell–Meiboom–Gill multi-pulse sequence; HSQC, heteronuclear single-quantum coherence; KSI, ketosteroid isomerase; 19-NTHS, 19-nortestosterone hemisuccinate; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; WALTZ, wideband alternating-phase low-power technique for zero residual splitting; WATERGATE, water suppression by gradient-tailored excitation; WT, wild-type
*Present address: The J. David Gladstone Institute, SF, CA94158, USA
Present address: 012 Fairchild Library, Division of Chemistry and Chemical Engineering, Caltech, Pasadena, CA91125, USA