title: Identification and characterization of a novel citrate synthase from Streptomyces diastaticus No. 7 strain M1033.
abstract: Citrate synthase (CS) is a key enzyme of the tricarboxylic acid cycle and is widely distributed among prokaryotes and eukaryotes. Here, we report for the first time, the cloning, expression, and characterization of a novel CS from Streptomyces diastaticus No. 7 strain M1033 (SdCS). Gel filtration chromatography and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analyses indicate that SdCS forms homodimers with a molecular mass of approximately 100.0 kDa. The predicted amino acid sequence of SdCS is highly similar to those of bacterial homodimeric type I CSs. The pH and temperature optima for SdCS activity were 8.0 and 35 °C, respectively. The half-life (t1/2 ) of SdCS was 10 Min at 50 °C and was increased to 210 Min in the presence of oxaloacetate. The kinetic parameters of SdCS (kcat = 262.8 and 230.7 s(-1); Km = 58.4 and 11.2 µM for acetyl-CoA and oxaloacetate, respectively) were comparable to those of dimeric CSs isolated from Gram-positive bacteria and eukaryotes. Moreover, SdCS activity was inhibited by ATP and ADP and stimulated by AMP. These findings provide a foundation for further investigations on the three-dimensional structure and mechanism of catalysis of SdCS.
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Yadong
authors.initials: Y
authors.lastname: Ge
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Ping
authors.initials: P
authors.lastname: Song
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002950:Citrate (si)-Synthase
keywords: Streptomyces diastaticus; Western blot; citrate synthase; enzymatic characterization; gel filtration chromatography; matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS)