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N-15 NMR relaxation studies of Y14F mutant of ketosteroid isomerase: The influence of mutation on backbone mobility

Title
N-15 NMR relaxation studies of Y14F mutant of ketosteroid isomerase: The influence of mutation on backbone mobility
Authors
Lee, HJYoon, YJJang, DSKim, CCha, HJHong, BHChoi, KYLee, HC
POSTECH Authors
Choi, KY
Date Issued
Aug-2008
Publisher
OXFORD UNIV PRESS
Abstract
The backbone dynamics of Y14F mutant of Delta(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 (15)N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S(2)) 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 S(2) values, whereas the S(2) 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.
Keywords
backbone dynamics; ketosteroid isomerase; mutant; NMR; relaxation; PUTIDA BIOTYPE-B; BOUND DELTA(5)-3-KETOSTEROID ISOMERASE; HYDROGEN-BOND NETWORK; DNA-BINDING DOMAIN; ACTIVE-SITE; PSEUDOMONAS-TESTOSTERONI; STAPHYLOCOCCAL NUCLEASE; DYNAMICS; SPECTROSCOPY; ENZYME
URI
http://oasis.postech.ac.kr/handle/2014.oak/28800
DOI
10.1093/JB/MVN053
ISSN
0021-924X
Article Type
Article
Citation
JOURNAL OF BIOCHEMISTRY, vol. 144, no. 2, page. 159 - 166, 2008-08
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최관용CHOI, KWAN YONG
Div of Integrative Biosci & Biotech
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