Peptidylglycine alpha amidating enzyme
Many neuropeptides and peptide hormones are posttranslationally modified by α-amidation, involving the conversion of a carboxy-terminal glycine residue into an amide. PHM is copper-, oxygen-, and ascorbate-dependent and hydroxylates the α-carbon of a glycine residue at the carboxyl terminus of the substrate peptide. c DNA clones corresponding to Am PAM-1 and Am PAM-2 were also obtained from a planula c DNA library (Brower et al.The presence of the amide moiety is often essential for the biological activity of the peptide (reviewed in Eipper et al. Familiar examples include oxytocin, vasopressin, and substance P. The resulting intermediate, peptidyl-α-hydroxyglycine, is dealkylated by the zinc-dependent enzyme PAL, releasing the final α-amidated peptide and glyoxalate. The finding of both bifunctional PAM and monofunctional PHM in the nematode indicates that separate and joint encoding of the two enzymes are not necessarily mutually exclusive within a species (Asada et al. Transcriptional and posttranslational regulation of these enzymes is complex. Splice variants, although less numerous, have been identified in other species including human, the frog (Glauder et al. 1997) using standard techniques (Sambrook and Russell 2001); these have been submitted to Gen Bank (accessions: JN790631–JN790632).1990; Bundgaard and Kahns 1991) but requires PAL activity in the acidic secretory vesicles where the reaction generally occurs in vertebrates (Eipper et al. Numbers to the left of the rat sequence indicate amino acid residue numbers in that sequence. Conserved cysteine residues C (the last of which is not shown) form structural disulphide bonds (Kolhekar et al. Dark gray shading indicates residues that are identical in more than half of the sequences; light gray shading indicates residues that are similar in more than half of the sequences. Dark gray shading indicates residues that are identical in more than half of the sequences; light gray shading indicates residues that are similar in more than half of the sequences. Some invertebrates, too, possess a gene encoding a bifunctional enzyme: the first to be reported was in the marine mollusc (Mair et al. Reactions were run on an ABI 3730 sequencer at the Biomolecular Resource Facility (John Curtin School of Medical Research, Australian National University). 1999) were made using the Clontech SMART c DNA Synthesis Kit, according to the manufacturer’s instructions, with RNA prepared from staged embryos, larvae, and adults.Here, we report sequence and expression data for two PAMs from the coral (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. Sequences which, when used as queries to search the Genbank nonredundant sequence database, had known PAMs as top hits were assembled with DNASTAR Seq Man (Lasergene), resulting in the identification of two predicted To verify the existence of the predicted Am PAM-1 transcript, primers were designed to match the 5′ and 3′ untranslated regions (UTRs).
The only enzymes known to catalyse the α-amidation reaction are the consecutively acting peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) (Bradbury et al. The first reaction is rate limiting in the production of amidated peptides (Mains et al. For example, the extensively studied rat PAM has at least seven splice variants across different tissues, including transcripts coding for soluble and membrane-bound PAM and for PHM alone (Eipper et al. Other transcripts allow alternative forms to be generated posttranslationally via the inclusion of proteolytic sites between the two domains, before the transmembrane domain, or before the cytosolic tail of membrane-bound PAM (Eipper et al. The cleaved cytosolic tail of PAM has been shown to localize to the nucleus and regulate transcription and perhaps ultimately secretory vesicle metabolism (Francone et al. Sequencing was performed using vector and internal primers with Big Dye Terminator v.1991); the second proceeds spontaneously under neutral to alkaline conditions (Tajima et al. Conserved residues for which structural and/or functional roles have been identified are indicated by dots. This polypeptide is sometimes cleaved posttranslationally into its two constituent parts, each of which can function independently. Stand-alone PHMs were also reported, and a separately encoded PAL posited, for another flatworm (Asada et al. 3.1 (Applied Bio Systems) according to manufacturer’s instructions.Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity.Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL).
In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification.Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. 2012) were searched using TBlast X and TBlast N and query amidating enzyme sequences from a closely related sequenced organism, the sea anemone (identified in Anctil 2009; accessions XM_001634152 and XM_001626445).