Enzyme Discovery for Natural Product Biosynthesis

Aim 1. Identify novel microbial natural product pathways and target the enzymes for structure determination by the PSI:Biology Network. These will be combinations of proteins of known function but unknown structure; unknown function and unknown structure; or where warranted, known fold family but unknown/ unmodelable active sites. We have nominated well-chosen representative targets to the PSI Network for X-ray structures of the enediyne polyketide synthase (PKS) and associated enzymes involved in the biosynthesis of the enediyne compounds of 10-membered (calicheamicin, dynemicicn and esperamicin) enediynes, indolocarbazole (rebeccamycin, staurosporine and AT2433), aromatic polyketide (hedamycin and pluramycin) and novel glycoside and S-adenosylmethionine precursors. In years 2-4, we will continue to nominate additional novel targets deriving from a new natural discovery effort launched as part of the new Center for Pharmaceutical Research and Innovation at the University of Kentucky under the directorship of Prof. Thorson. This new effort includes natural product discovery from novel microbes found within unique ecological niches across the Commonwealth coupled with genome sequencing to rapidly identify target gene clusters as a starting point for subsequent biochemical and structural studies. Aim 2. Conduct experimental functional studies on the set of enzymes from Aim 1. The second aim is designed to use enzyme structural determination as a tool to help understand the intricacies of enzyme specificity and catalysis in natural product biosynthesis. We will use biochemical and biophysical methods to study, for example, mechanisms of glycosyltransferases we have specifically engineered or evolved to engender novel properties - mechanistic (N- versus C- O-glycosylation), thermodynamic ('reversible' versus 'nonreversible') and substrate tolerance (broad substrate specificity versus stringent) - in an attempt to establish the structural determinates that dictate these important properties. In a similar manner, we will expand the scope of targets relevant to our chemoenzymatic methods to also include engineered/evolved Sadenosylmethionine synthetases and methyltransferases. In addition, we will continue to provide biochemical support for the structures emerging from aim 1 with prioritization based upon mechanistic and/or structural novelty. Aim 3. Better annotate new pathways on the basis of new structures using bioinformatics approaches. The information from structure determinations of natural product enzymes and their homologs will be used to provide additional support and improve functional annotations for other natural product biosynthetic pathways. This is possible because of the general conservation of folds with their reaction classes and the recognition that there are models for elaboration of pathways from gene duplication and/or recruitment. Specifically, we will use information gleaned from aims 1 and 2 to help advance our annotation efforts of new gene clusters identified as part of the CPRI natural product discovery effort briefly mentioned under aim 1.