Structural Biology of Complex Enzymes

University Of Michigan At Ann Arbor, Ann Arbor MI

Investigators

Abstract

Project Summary Natural product biosynthesis encompasses a wealth of interesting chemical transformations. The corresponding enzymes have tremendous biocatalytic potential within or outside their natural pathways. Realizing this potential requires a structure-based understanding of enzyme function as well as substrate selectivity and range. Three enzyme systems will be investigated in this project. Thioesterases (TEs) form the macrolactone scaffolds of macrolide antibiotics by cyclizing linear polyketides during offloading from carrier proteins. The TEs are potential biocatalysts to diversify the macrolide repertoire in the ongoing battle against antibiotic resistance. However, TEs generally cyclize only their native substrates and offload linear products for non-native substrates. How several TEs direct macrocycle formation will be determined by visualizing covalent intermediates trapped by an unnatural amino acid substituted for the TE catalytic nucleophile. The structures together with assay data from a panel of substrates and products will provide a wealth of information on the determinants of macrocycle vs. linear product formation and will inform efforts to engineer macrocycle diversity. The second enzyme is a putative terminal amidation domain (TAD) that forms linear amide products during offloading from carrier proteins in several nonribosomal peptide synthetase (NRPS) pathways. ORFs encoding TADs are widely distributed in the bacterial kingdom, located predominantly at the end of NRPS gene clusters. Building on the results developed during the current funding period – a comprehensive database of TAD sequences, biochemical data and crystal structures – we will test the hypotheses that TAD catalyzes amidation and that nitrogen is derived by oxidative release or amide hydrolysis. Identification of the unknown nitrogen source could enhance the potential of TADs as general amidation catalysts. The third enzyme system is the BURP cyclases that form burpitides, a new class of ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products in plants. The recently discovered Cu- and O2-dependent BURP cyclases use a novel Cu center to form crosslinks in short core peptides prior to proteolytic release of the mature burpitides. Building on our initial crystal structure, this project will enhance the understanding of BURP function and structure. We will expand the structure database of BURP cyclases, visualize a fully formed Cu center, see how substrates engage with the BURP cyclase active site, investigate possibilities for substrate alteration by mutagenesis, and learn how proteases release cyclized burpitides and not their linear precursors. Each of the three enzyme systems has potential for several applications that will be advanced by the results of this project.