Heterologous expression and characterization of soluble recombinant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Actinosynnema pretiosum ssp. auranticum ATCC31565 through co-expression with Chaperones in Escherichia coli.
3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS), (EC 2.5.1.54) catalyzes the first step of the shikimate pathway, the route for the biosynthesis of aromatic compounds in plants and microbes. In Actinosynnema pretiosum, the aroF gene (GenBank: AF056968.1) encodes DAHPS to condensate phosphoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to generate DAHP. In this study, a recombinant pET28a-aroF plasmid was constructed and A. pretiosum DAHPS was successfully expressed in soluble form by co-expression with chaperonins GroEL/GroES in Escherichia coli. The purification and kinetic characterization of the expressed protein were then investigated. The DAHPS originated from A. pretiosum demonstrated a pronounced substrate inhibition by PEP but was not sensitive to E4P. The purified enzyme was completely inactivated by EDTA but potently activated by several bivalent metal ions, especially Mn(2+) and Co(2+).
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Na
N
Ma
Liujing
L
Wei
Yuxiang
Y
Fan
Qiang
Q
Hua
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002413:Cations, Divalent / Q000737:chemistry; D018835:Chaperonin 10 / Q000096:biosynthesis*; D018834:Chaperonin 60 / Q000096:biosynthesis*; D002614:Chelating Agents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D001426:Bacterial Proteins; D002413:Cations, Divalent; D018835:Chaperonin 10; D018834:Chaperonin 60; D002614:Chelating Agents; D011994:Recombinant Proteins; D004492:Edetic Acid; D010723:3-Deoxy-7-Phosphoheptulonate Synthase