title: Tailoring enzymes acting on carrier protein-tethered substrates in natural product biosynthesis.
abstract: Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered substrates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-β-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product biosynthesis and structural diversification. The ultimate goal is to use the in vitro findings to guide in vivo engineering of designer natural products.
authors.affiliation: The State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China.
authors.forename: Shuangjun
authors.initials: S
authors.lastname: Lin
authors.forename: Tingting
authors.initials: T
authors.lastname: Huang
authors.forename: Ben
authors.initials: B
authors.lastname: Shen
mesh_terms: D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry; D000642:Ammonia-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D001688:Biological Products / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D053281:Enediynes / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D054879:Halogenation; D006900:Hydroxylation; D010453:Peptide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D000617:Aminoglycosides; D000903:Antibiotics, Antineoplastic; D001426:Bacterial Proteins; D001688:Biological Products; D002352:Carrier Proteins; D053281:Enediynes; C058024:C 1027; D014443:Tyrosine; D000642:Ammonia-Lyases; D010453:Peptide Synthases