title: Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase.
abstract: Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.
authors.affiliation: Pohang Accelerator Laboratory, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea.
authors.forename: Hyung Jin
authors.initials: HJ
authors.lastname: Cha
authors.affiliation: Department of Life Sciences, POSTECH, Pohang, Republic of Korea.
authors.forename: Do Soo
authors.initials: do S
authors.lastname: Jang
authors.affiliation: Pohang Accelerator Laboratory, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea.
authors.forename: Jae-Hee
authors.initials: JH
authors.lastname: Jeong
authors.affiliation: Department of Life Sciences, POSTECH, Pohang, Republic of Korea.
authors.forename: Bee Hak
authors.initials: BH
authors.lastname: Hong
authors.affiliation: Department of Life Sciences, POSTECH, Pohang, Republic of Korea.
authors.forename: Young Sung
authors.initials: YS
authors.lastname: Yun
authors.affiliation: Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
authors.forename: Eun Ju
authors.initials: EJ
authors.lastname: Shin
authors.affiliation: Department of Life Sciences, POSTECH, Pohang, Republic of Korea. Electronic address: kchoi@postech.ac.kr.
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007664:Ketosteroids / Q000378:metabolism*; D008715:Methionine / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013251:Steroid Isomerases / Q000235:genetics*; D044366:Transition Temperature
chemical_list: D007664:Ketosteroids; D008715:Methionine; D013251:Steroid Isomerases
keywords: Enzyme catalysis; Ketosteroid isomerase; Met112; Stability; Structural analysis