TY - JOUR
T1 - Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin
AU - Sánchez, César
AU - Du, Liangcheng
AU - Edwards, Daniel J.
AU - Toney, Michael D.
AU - Shen, Ben
N1 - Funding Information:
We thank Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan, for providing S. atroolivaceus and Prof. Dr. W.F. Fleck, Hans-Knöll-Institut für Naturstoff-Forschung, Jena, Germany, for providing S. albus JA 3453. This work was supported in part by an Institutional Research Grant from the American Cancer Society and the School of Medicine, University of California, Davis; the National Institutes of Health Grant AI40475; and the Searle Scholars Program/The Chicago Community Trust. C.S. was supported in part by a postdoctoral fellowship from the Spanish Ministry of Education and Culture. D.E. was supported in part by a NIH Training Grant GM07377.
PY - 2001
Y1 - 2001
N2 - Background: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4′-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. Results: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (kcat/Km) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. Conclusions: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.
AB - Background: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4′-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. Results: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (kcat/Km) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. Conclusions: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.
KW - Acyl carrier protein
KW - Bleomycin
KW - Peptidyl carrier protein
KW - Phosphopantetheinyl transferase
KW - Streptomyces verticillus
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U2 - 10.1016/S1074-5521(01)00047-3
DO - 10.1016/S1074-5521(01)00047-3
M3 - Article
C2 - 11451672
AN - SCOPUS:0034930478
SN - 1074-5521
VL - 8
SP - 725
EP - 738
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 7
ER -