Peptidyl α-hydroxyglycine α-amidating lyases
The present patent application concerns an enzyme capable of catalysing the conversion of a α-hydroxyglycine to an α-amide and the use of such enzymes for producing a C-terminal α-amidated peptide.
1. A method for producing an α-amidated peptide comprising:
(i) allowing a target peptide with a C-terminal glycine residue to react with both a peptidylglycine alpha-hydroxylating monooxygenase (PHM) and a polypeptide consisting of amino acids 2-306 of SEQ ID NO: 1, amino acids 3-336 of SEQ ID NO: 2, amino acids 3-305 of SEQ ID NO: 3, or amino acids 3-279 of SEQ ID NO: 4, wherein said polypeptide has peptidyl-α-hydroxyglycine alpha-amidating lyase (PAL) activity under conditions suitable for enzymatic activity, and optionally further comprises 1-30 substitution(s) or deletion(s), and wherein the reaction with said PHM and said polypeptide on said target peptide is performed either in two separate steps or simultaneously; and
(ii) recovering the C-terminally α-amidated peptide.
2. The method of claim 1 , wherein the polypeptide has been expressed in a recombinant E. coli.
3. The method of claim 1 , wherein the polypeptide from a prokaryotic organism has an amino acid sequence comprising 0, 1 or 2 cysteine (Cys) residues.
4. The method of claim 1 , wherein the polypeptide is in a catalytically active form and does not require a step of refolding for obtaining catalytic activity.
5. The method of claim 1 , wherein the target peptide is selected from the group consisting of amylin, Neuropeptide Y (NPY), Peptide YY (PYY), PYY-3-36, Pancreatic polypeptide (PP), Glucagon like peptide (GLP-1), gastrin, calcitonin, calcitonin related peptide (CGRP), gastrin releasing peptide, vasopressin, oxytocin, neurokinin A, secretin, pancreastatin, pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N) or functional analogs thereof.