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Biochemical and structural characterization of 3‐ketosteroid‐Δ 1 ‐dehydrogenase, a candidate enzyme for efficient bioconversion of steroids
Author(s) -
Mao Shuhong,
Guo Qianqian,
Xu Panpan,
Gao Dengke,
Cheng Xiaotao,
Zhu Menglu,
Sun Dengyue,
Zhu Zhangliang,
Lu Fuping,
Qin HuiMin
Publication year - 2019
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.5780
Subject(s) - ketosteroid , bioconversion , dehydrogenase , cofactor , enzyme , escherichia coli , chemistry , biochemistry , isomerase , stereochemistry , substrate (aquarium) , arthrobacter , biology , ecology , fermentation , gene
Background 3‐ketosteroid‐Δ 1 ‐dehydrogenase, a key enzyme involved in microbial catabolism of sterols, catalyzes the 1, 2‐desaturation of steroidal substrates using FAD as a cofactor. Recombinant 3‐ketosteroid‐Δ 1 ‐dehydrogenase from Arthrobacter simplex (KsdD4) was expressed in Escherichia coli BL21 (DE3). Results KsdD4 exhibited optimal activity at pH 7.0 and 35°C. KsdD4 had the highest activity toward 4‐androstene‐3,17‐dione (AD) and a moderate activity toward cortisone acetate and hydrocortisone. Structure‐based homology modeling revealed that Y118, Y355, Y528 and G532 are located in the active site, thus the most likely catalytic residues. The substrate‐binding cavity was composed of hydrophobic residues with small side chains, including A49, V328, A390 and A531, which facilitate the recognition of steroidal substrates with large C17 side‐chains. E332 forms a unique hydrogen bond with substrates. Recombinant E. coli resting cells expressing KsdD4 showed high catalytic activity in the presence of various organic solvents. A fed‐batch bioconversion using resting cells resulted in efficient production of 2.66 g L −1 androst‐1,4‐diene‐3,17‐dione (ADD). Conclusion Biochemical data and structural analysis greatly advanced our understanding of the KsdD family enzymes, and the fed‐batch strategy improved the bioconversion of steroids. © 2018 Society of Chemical Industry