Chapter 10 using phosphopantetheinyl transferases for enzyme posttranslational activation, site specific protein labeling and identification of natural product biosynthetic gene clusters from bacterial genomes.
Phosphopantetheinyl transferases (PPTases) covalently attach the phosphopantetheinyl group derived from coenzyme A (CoA) to acyl carrier proteins or peptidyl carrier proteins as part of the enzymatic assembly lines of fatty acid synthases (FAS), polyketide synthases (PKS), and nonribosomal peptide synthetases (NRPS). PPTases have demonstrated broad substrate specificities for cross-species modification of carrier proteins embedded in PKS or NRPS modules. PPTase Sfp from Bacillus subtilis and AcpS from Escherichia coli also transfer small molecules of diverse structures from their CoA conjugates to the carrier proteins. Short peptide tags have thus been developed as efficient substrates of Sfp and AcpS for site-specific labeling of the peptide-tagged fusion proteins with biotin or organic fluorophores. This chapter discusses the use of PPTases for in vivo and in vitro modification of PKS and NRPS enzymes and for site-specific protein labeling. We also describe a phage selection method based on PPTase-catalyzed carrier protein modification for the identification of PKS or NRPS genes from bacterial genomes.
Department of Chemistry, The University of Chicago, Chicago, Illinois, USA.
Murat
M
Sunbul
Keya
K
Zhang
Jun
J
Yin
D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001688:Biological Products / Q000096:biosynthesis* / Q000235:genetics; D003065:Coenzyme A / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D016680:Genome, Bacterial / Q000235:genetics*; D005810:Multigene Family / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D019151:Peptide Library; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*
D001426:Bacterial Proteins; D001688:Biological Products; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D019151:Peptide Library; C082662:phosphopantetheinyl transferase; C073125:acpS protein, E coli; D048630:Polyketide Synthases; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D003065:Coenzyme A