TY - JOUR
T1 - In Vivo and In Vitro Trans-Acylation by BryP, the Putative Bryostatin Pathway Acyltransferase Derived from an Uncultured Marine Symbiont
AU - Lopanik, Nicole B.
AU - Shields, Jennifer A.
AU - Buchholz, Tonia J.
AU - Rath, Christopher M.
AU - Hothersall, Joanne
AU - Haygood, Margo G.
AU - Håkansson, Kristina
AU - Thomas, Christopher M.
AU - Sherman, David H.
N1 - Funding Information:
We thank Niels Lindquist for providing sampling and laboratory support, and W. Clay Brown and Jim DelProposto at the LSI High Throughput Protein core facility for generating and screening the BryP didomain and AT 2 overexpression constructs, and for supplying the pRARE-CDF coexpression plasmid. We would like to thank Jeff Kittendorf for cloning the EryAIII M6 construct, Brian Beck for cloning the PikAIV M6 WT and mutant constructs, and Sabine Grüschow for cloning the sfp coexpression vector, pSG701. Sylvie Garneau-Tsodikova generously provided purified apo and holo preparations of CloN5. N.B.L was supported by National Institutes of Health (NIH) National Research Service Award fellowship 5F32CA110636. Research support was generously provided by NIH grant R01 GM076477 and the Hans W. Vahlteich Professorship to D.H.S.
PY - 2008/11/24
Y1 - 2008/11/24
N2 - The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete open reading frame (ORF) (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.
AB - The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete open reading frame (ORF) (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.
KW - CHEMBIO
UR - http://www.scopus.com/inward/record.url?scp=56049123651&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=56049123651&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2008.09.013
DO - 10.1016/j.chembiol.2008.09.013
M3 - Article
C2 - 19022178
AN - SCOPUS:56049123651
SN - 1074-5521
VL - 15
SP - 1175
EP - 1186
JO - Chemistry and Biology
JF - Chemistry and Biology
IS - 11
ER -