title: Characterization and expression of AMP-forming Acetyl-CoA Synthetase from Dunaliella tertiolecta and its response to nitrogen starvation stress.
abstract: AMP-forming acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-CoA. Here, a cDNA of ACS from Dunaliella tertiolecta (DtACS) was isolated using RACEs. The full-length DtACS cDNA (GenBank: KT692941) is 2,464 bp with a putative ORF of 2,184 bp, which encodes 727 amino acids with a predicted molecular weight of 79.72 kDa. DtACS has a close relationship with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. ACSs existing in Bacteria, Archaea and Eukaryota share ten conserved motifs (A1-A10) and three signature motifs (I-III) of the acyl-adenylate/thioester forming enzyme superfamily. DtACS was expressed in E. coli BL21 as Trx-His-tagged fusion protein (~100 kDa) and the enzymatic activity was detected. The recombinant DtACS was purified by HisTrap(TM) HP affinity chromatography to obtain a specific activity of 52.873 U/mg with a yield of 56.26%, which approached the specific activity of ACS isolated from other eukaryotes. Kinetic analysis indicated that the Km of DtACS was 3.59 mM for potassium acetate, and the purified DtACS exhibited a temperature optimum of 37 °C and a pH optimum of 8.0. In addition, the expression levels of DtACS were increased after nitrogen starvation cultivation, indicating that ACS activity may be related to the lipid accumulation under nitrogen deficient condition.
authors.affiliation: College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
authors.forename: Ming-Hua
authors.initials: MH
authors.lastname: Liang
authors.affiliation: College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
authors.forename: Xiao-Ying
authors.initials: XY
authors.lastname: Qv
authors.affiliation: College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
authors.forename: Hong-Hao
authors.initials: HH
authors.lastname: Jin
authors.affiliation: College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Jiang
mesh_terms: D000106:Acetate-CoA Ligase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D000460:Chlorophyta / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D020418:Algal Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D000106:Acetate-CoA Ligase; D009584:Nitrogen