title: 15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility.
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.
authors.affiliation: Department of Chemistry, and Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang, 790-784 Republic of Korea.
authors.forename: Hyeong Ju
authors.initials: HJ
authors.lastname: Lee
authors.forename: Ye Jeong
authors.initials: YJ
authors.lastname: Yoon
authors.forename: Do Soo
authors.initials: DS
authors.lastname: Jang
authors.forename: Chul
authors.initials: C
authors.lastname: Kim
authors.forename: Hyung Jin
authors.initials: HJ
authors.lastname: Cha
authors.forename: Bee Hak
authors.initials: BH
authors.lastname: Hong
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
authors.forename: Hee Cheon
authors.initials: HC
authors.lastname: Lee
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001665:Binding Sites; D020604:Comamonas testosteroni / Q000201:enzymology; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009154:Mutation; D009277:Nandrolone / Q000031:analogs & derivatives / Q000737:chemistry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D013251:Steroid Isomerases / Q000737:chemistry* / Q000235:genetics*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009587:Nitrogen Isotopes; D014443:Tyrosine; C068109:19-nortestosterone 17-hemisuccinate; D009277:Nandrolone; D013251:Steroid Isomerases; C022660:steroid delta-isomerase