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

Cnidarians are primitive animals that use neuropeptides as their transmitters. All the numerous cnidarian neuropeptides isolated, so far, have a carboxy-terminal amide group that is essential for their actions. This strongly suggests that alpha-amidating enzymes are essential for the functioning of primitive nervous systems. In mammals, peptide amidation is catalyzed by two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) that act sequentially. These two activities are contained within one bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), which is coded for by a single gene. In a previous paper (F. Hauser et al., Biochem. Biophys. Res. Commun. 241, 509-512, 1997) we have cloned the first known cnidarian PHM from the sea anemone Calliactis parasitica. In the present paper we have determined the structure of its gene (CP1). CP1 is >12 kb in size and contains 15 exons 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 mammals. 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, whereas both splicing variants have around 42% sequence identity with the PHM part of rat PAM. Essential amino acid residues for the catalytic activity and 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 and have the same intron phasing as eight introns in the rat PAM gene, showing that the sea anemone PHM is not only structurally, but also evolutionarily related to the PHM part of rat PAM. Copyright 2000 Academic Press.