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

Journal of Biochemistry
Hyeong Ju LeeHee Cheon Lee

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 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.

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Citations

Jun 7, 2013·Molecules and Cells·Hyung Jin ChaKwan Yong Choi
Mar 8, 2012·PLoS Computational Biology·Deeptak VermaDennis R Livesay
Nov 9, 2010·The Journal of Steroid Biochemistry and Molecular Biology·Masae HorinouchiToshiaki Kudo
Jul 5, 2016·Biochimica Et Biophysica Acta·Hyung Jin ChaKwan Yong Choi

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