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Mark Zabriskie
Professor, College of Pharmacy
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RESEARCH:
Research in this lab focuses on applying the combined techniques of organic chemistry, biochemistry and molecular biology to study enzymes of biomedical importance. Systems being investigated range from therapeutic targets to enzymes involved in the assembly of bioactive natural products. All of our projects center on enzymes that utilize amino acids as substrates.
One group of projects focuses on studying the mechanisms and developing inhibitors of amino acid-degrading enzymes in the central nervous system. By developing agents that affect levels of specific neuroactive intermediates in these pathways we hope to produce useful pharmacological tools for deciphering the central roles of these metabolites. Of primary interest is the CNS-specific metabolism of lysine via pipecolic acid, a metabolite believed to enhance inhibitory neurotransmission by interacting with the GABAA receptor. Specific inhibitors of the flavoenzyme that degrades pipecolic acid may result in a significant increase in the synaptic concentration of pipecolic acid with a concomitant increase in its neurological effects and serve as leads to new anticonvulsants. Much of this work involves the synthesis and kinetic evaluation of substrate analogs designed to be mechanism-based inactivators. These irreversible enzyme inhibitors often form covalent attachments with the enzyme or cofactor and provide important information on the nature of the active site and the exact chemical mechanism of the target enzyme.
A second area of interest is the molecular genetics and enzymology of bioactive natural product biosynthesis. We are presently studying the biosynthesis of peptides such as the antituberculosis agent capreomycin and the peptide-nucleoside antifungal agent blasticidin S. This work entails cloning and characterizing self resistance and biosynthetic genes from a producing organism, usually a Streptomyces species, and then expressing individual genes or entire pathways in a surrogate host.
We are particularly interested in the nonribosomal peptide synthetases (NRPS) that function in these pathways. These large, multifunctional proteins catalyze the sequence-specific formation of small peptides without using a nucleic acid template. A single NRPS module contains individual domains that activate a specific amino acid as the acyladenylate, carry out modifications such as N-methylation or epimerization, and catalyze the formation of a peptide bond with an amino acid from another module. Frequently peptide synthetases utilize nonprotein amino acids as substrates and through our studies we hope to understand factors governing substrate specificity so that these proteins can be modified to accommodate a wider array of amino acid precursors. The aim is to use these modular systems to reengineer the biosynthetic machinery and produce novel bioactive compounds.
Recently we have extended these studies to include cloning peptide synthetase genes from unculturable marine organisms. Numerous unusual amino acids are found in peptides of marine origin, often from invertebrates or microbes that can not be cultured in the laboratory. Accessing these NRPS genes will greatly expand the number of unique adenylation domains available for combinatorial biosynthetic approaches to generate molecular diversity and produce new bioactive peptides.