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

The backbone dynamics of Y14F mutant of Δ5-3-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 sho...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2008-08, Vol.144 (2), p.159-166
Main Authors: Lee, Hyeong Ju, Yoon, Ye Jeong, Jang, Do Soo, Kim, Chul, Cha, Hyung Jin, Hong, Bee Hak, Choi, Kwan Yong, Lee, Hee Cheon
Format: Article
Language:English
Subjects:
Amino Acid Substitution
backbone dynamics
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Binding Sites
Comamonas testosteroni - enzymology
Hydrogen Bonding
ketosteroid isomerase
Models, Molecular
mutant
Mutation
Nandrolone - analogs & derivatives
Nandrolone - chemistry
Nitrogen Isotopes
NMR
Nuclear Magnetic Resonance, Biomolecular
relaxation
Steroid Isomerases - chemistry
Steroid Isomerases - genetics
Tyrosine - chemistry
Tyrosine - genetics
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Summary:The backbone dynamics of Y14F mutant of Δ5-3-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.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvn053