Premium
Alteration of the substrate specificity of ketosteroid isomerase from Pseudomonas putida Biotype B
Author(s) -
Cha Hyung Jin,
Jang Do Soo,
Shin Han Seop,
Choi Kwan Yong
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1007.1
Subject(s) - ketosteroid , isomerase , substrate (aquarium) , mutant , substrate specificity , chemistry , pseudomonas putida , steroid , mutagenesis , biochemistry , stereochemistry , enzyme , isomerization , protein engineering , catalysis , biology , gene , hormone , ecology
Most enzymes are quite specific for their natural substrates. Therefore, it is often necessary to modify or broaden their substrate selectivity for industrial or biotechnology applications. However, there are many difficulties to change the substrate specificity, since the relationship between substrate specificity and structure is too complex to understand. Especially, there are few examples that report changing the substrate specificity in steroid‐binding proteins by mutagenesis. Utilizing a ketosteroid isomerase (KSI) from bacterial source, we studied about the substrate specificity. We focused on role of the end of steroid–binding cleft, which interact with D‐ring derivative steroids. Therefore, we made following mutants such as M90A, M90G, and G60L, to alter the substrate specificity by broadening or narrowing this region. KSI catalyzes the isomerization of various Δ 5 ‐3‐Ketosteroid to Δ 4 ‐3‐Ketosteroid. Steroids such as 5‐Androsten‐3,17‐dione, 5(10)‐estren‐3,17‐dione, and 5‐pregnene‐3,20‐dione was used as the substrate. We found that the catalytic activity of M90A mutant against 5‐Pregnene‐3,20‐dione was increased about 2.5‐fold relative to that of WT.