Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli.
O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.
Zhao
Chunhui
C
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Japan.
Kumada
Yoichi
Y
Imanaka
Hiroyuki
H
Imamura
Koreyoshi
K
Nakanishi
Kazuhiro
K
D003541:Cystathionine beta-Synthase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003547:Cysteine Synthase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology
D029968:Escherichia coli Proteins; D003547:Cysteine Synthase; C513541:cysM protein, E coli; D003541:Cystathionine beta-Synthase