title: Expression, purification and functional characterization of a novel 3α-hydroxysteroid dehydrogenase from Pseudomonas aeruginosa.
abstract: 3α-Hydroxysteroid dehydrogenase (3α-HSD) catalyzes the oxidation of the 3-hydroxyl group of steroids. The enzymatic conversion is a critical step in the enzymatic assay of urinary sulfated bile acids (SBAs), which is a valuable diagnosis index of hepatobiliary diseases. However, the source of 3α-HSD for clinical applications is limited. In this study, an open reading frame (ORF) encoding a novel 3α-HSD was successfully cloned from Pseudomonas aeruginosa and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified by immobilized metal ion affinity chromatography. Enzyme characterization studies revealed that the protein has 3α-HSD activity and the Km value for sodium cholate is 1.06 mmol L(-1). More than 60% relative enzyme activity was observed in a wide range of pH and temperature, with an optimum pH at 8.0 and an optimum temperature at 30°C. The enzyme's good thermostability under 40°C would be favorable in clinical applications. Ion interference experiments indicated that Zn(2+) was an activating cofactor which increased the enzyme activity 1.75-fold. With the favorable characteristics mentioned above, the new 3α-HSD is a promising enzyme for clinical applications. More importantly, the present work is the first report on a 3α-HSD from P. aeruginosa.
authors.affiliation: West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China; School of Basic Medical Sciences, Chengdu Medical College, Chengdu 610500, China. Electronic address: chenjmorange@163.com.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Chen
authors.affiliation: West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. Electronic address: xiufengg@163.com.
authors.forename: Xiufeng
authors.initials: X
authors.lastname: Gao
authors.affiliation: West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Hong
authors.affiliation: West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
authors.forename: Liting
authors.initials: L
authors.lastname: Ma
authors.affiliation: School of Chemical Engineering, Sichuan University, Chengdu 610065, China. Electronic address: lysgxf2005@qq.com.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Li
mesh_terms: D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D019216:Metals, Heavy; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D011994:Recombinant Proteins; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
keywords: 3α-Hydroxysteroid dehydrogenase; P. aeruginosa; Short chain dehydrogenase/reductase; Sulfated bile acid