title: Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus.
abstract: Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells.
authors.affiliation: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.
authors.forename: Sabah
authors.initials: S
authors.lastname: Ul-Hasan
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Burgess
authors.forename: Joanna
authors.initials: J
authors.lastname: Gajewiak
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Hao
authors.initials: H
authors.lastname: Hu
authors.forename: Mark
authors.initials: M
authors.lastname: Yandell
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.forename: Pradip K
authors.initials: PK
authors.lastname: Bandyopadhyay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D020916:Conotoxins / Q000737:chemistry*; D052078:Conus Snail / Q000737:chemistry*; D004330:Drosophila / Q000166:cytology / Q000235:genetics; D015723:Gene Library; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D020916:Conotoxins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase
keywords: Conotoxins; Peptidylglycine α-amidating monooxygenase; Posttranslational modification