5G2G

Crystal structure of ketosteroid isomerase containing M116K mutation in the equilenin-bound form

Summary for 5G2G

• Entry DOI: 10.2210/pdb5g2g/pdb
• Descriptor: STEROID DELTA-ISOMERASE, EQUILENIN (3 entities in total)
• Functional Keywords: isomerase, ketosteroid isomerase, catalysis, stability, structural analysis, conserved met112
• Biological source: PSEUDOMONAS PUTIDA
• Total number of polymer chains: 2
• Total formula weight: 28654.53

Authors

Cha, H.J.,Jeong, J.H. (deposition date: 2016-04-08, release date: 2016-07-13, Last modification date: 2024-01-10)

Primary citation

Cha, H.J.,Jang, D.S.,Jeong, J.H.,Hong, B.H.,Yun, Y.S.,Shin, E.J.,Choi, K.Y.
Role of Conserved met112 Residue in the Catalytic Activity and Stability of Ketosteroid Isomerase.
Biochim.Biophys.Acta, 1864:1322-, 2016

PubMed 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.

PubMed: 27375051
DOI: 10.1016/J.BBAPAP.2016.06.016

Experimental method

X-RAY DIFFRACTION (1.599 Å)