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Show COLLEGE OF SCIENCE UNDERGRADUATE RESEARCH ABSTRACTS PEPTIDYLGLYCINE a-AMIDATING MONOOXYGENASE (PAM) IN C. GEOGRAPHUS Daniel Burgess (Baldomera Olivera, Pradip Bandyopadhyay) Department of Biology University of Utah Many proteins in animals undergo post-translational modifications to become biologically active. In most eukaryotes, the bifunctional enzyme responsible for the amidation of peptides and proteins containing a C-terminal glycine is called peptidylglycine a-amidating monooxygenase (PAM); PAM contains two active domains called peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), respectively (See Figure 1). Several conopeptides produced in the venom duct of cone snails paralyze prey by targeting particular ion channels and neurotransmitter receptors with high specificity; due to this high targeting specificity, conopeptides are of medicinal interest. Many of these conopeptides undergo C-terminal amidation in order to become biologically active in the venom duct. Therefore, PAM in the Conus genus should be studied to optimize the use of the PAM enzyme in the synthesis of amidated conopeptides. A complete ~2.4 kb cDNA sequence encoding for the complete Conus geographus PAM was assembled from overlaps in 3'RACE, 5'RACE, and transcriptome sequences. W h e n evaluating PCR product sequences, three isoforms were identified. The complete geographus PAM sequences were aligned with known PAM sequences in A. californica, R. norvegicus, C. bullatus, and D. melanogaster (PAM domains are separate in Drosophila.). Using this alignment as the basis of our analysis, isoform 2 contains all crucial amino acids to perform the full PAM function; however, both isoform 1 and 3 are expected to be unable to perform the PAL function due to missing residues (See Figure 2). It is speculated the isoforms lacking a PAL function may work on a specific substrate. Further analysis of these sequences in C. bullatus and C. geographus indicate they are 83.7% identical at the amino acid level and 89.5% at the level of nucleotides. This degree of homology is consistent with the observation that the genes C01, 12s, and 16s in the two species are 79.5%, 88.7% and 85.5% identical respectively. Further experiments in progress include mapping the PHM and PAL domains of the enzyme and the enzymatic activity of the enzyme on conopeptides. Daniel Burgess Baldomera Olivera Pradip Bandyopadhyay |