Gene cloning, expression and functional characterization of a phosphopantetheinyl transferase from Vibrio anguillarum serotype O1.
Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism. Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases). Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V. anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid. In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V. anguillarum serotype O1. A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase. angD was overexpressed in E. coli and the resultant protein, AngD, was purified. Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E. coli and purified. The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture. Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E. coli, modifying PCP and ArCP completely.
Liu
Qin
Q
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Ma
Yue
Y
Zhou
Lingyun
L
Zhang
Yuanxing
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups)