Modular Enzymatic Assembly Lines for Antibiotics

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DESCRIPTION (provided by applicant):Nature assembles thousands of polyketide (PK) and nonribosomal peptide (NRP) natural product antibiotics on multimodular enzymatic assembly lines. Growing chains are tethered as a series of elongating covalent acyl-S-protein intermediates, attached by phosphopantetheinyl chains to carrier protein domains, typically one per module. This proposal examines catalytic machinery and chemical logic for two sets of maturation steps in antibiotic natural products. Nascent scaffolds are subject to tailoring reactions by dedicated enzymes that often are crucial for maturation of target biological activities. In Specific Aim 1 we prose examination of scope and mechanism of two types of oxidative tailoring enzymes for NRP and NR-PK hybrids: halogenases and oxygenases. Tailoring halogenases use either FADH2 or mononuclear nonheme FeII, dependent on electronic demand of the carbon site in the antibiotic. We plan to use halogenase gene probes to clone biosynthetic gene clusters, e.g. for kutzneride, and examine scope of the halogenative tailoring. We shall also examine the activity and mechanism of tailoring oxygenases that act in antibiotic scaffold maturations. Specific Aim 2 addresses catalytic domains in assembly lines involved in novel chemistry during chain elongation and termination steps. These include ester backbone linkages in place of amides, the recruitment of transglutaminase homologs as condensation domains that act in trans, formation of C-terminal pyrrolidinedione rings, and reductive chain termination steps.