Genomic organization and splicing variants of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

Cnidarians are primitive animals that use neuropeptides as their transmitte rs. All the numerous cnidarian neuropeptides isolated, so far, have a carbo xy-terminal amide group that is essential for their actions. This strongly suggests that rr-amidating enzymes are essential for the functioning of pri mitive nervous systems. In mammals, peptide amidation is catalyzed by two e nzymes, peptidylglycine alpha -hydroxylating monooxygenase (PHM) and peptid yl-alpha -hydroxyglycine alpha -amidating lyase (PAL) that act sequentially . These two activities are contained within one bifunctional enzyme, peptid ylglycine alpha -amidating monooxygenase (PAM), which is coded for by a sin gle gene. In a previous paper (F. Hauser et at, Biochem. Biophys. Res. Comm un. 241, 509-512, 1997) we have cloned the first known cnidarian PHM from t he sea anemone Calliactis parasitica. In the present paper we have determin ed the structure of its gene (CP1). CP1 is >12 kb in size and contains 15 e xons and 14 introns. The last coding exon (exon 15) contains a stop codon, leaving no room for PAL and, thereby, for a bifunctional PAM enzyme as in m ammals. Furthermore, we found a CP1 splicing variant (CP1-B) that contains exon-9 instead of exon-8, which was present in the previously characterized PHM cDNA (CP1-A). CP1-A and -B have 97% amino acid sequence identity, wher eas both splicing variants have around 42% sequence identity with the PHM p art of rat PAM. Essential amino acid residues for the catalytic activity an d the 3D structure of PHM are conserved between CP1-A, -B and the PHM part of rat PAM. Furthermore, eight introns in CP1 occur in the same positions a nd have the same intron phasing as eight introns in the rat PAM gene, showi ng that the sea anemone PHM is not only structurally, but also evolutionari ly related to the PHM part of rat PAM. (C) 2000 Academic Press.