Novel peptidyl alpha-hydroxyglycine alpha-amidating lyases

Abstract

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 . An isolated polypeptide capable of catalysing the conversion of a α-hydroxyglycine to an α-amide, wherein said polypeptide comprises an amino acid sequence comprising the following motif 1:
 Xaa 1  Val Xaa 2  Asp Arg Xaa 3  Xaa 4  Xaa 5  Arg Xaa 6  Gln Xaa 7  Xaa 8  Xaa 9  Xaa 10  Xaa 11  Gly Xaa 12  Xaa 13  Xaa 14  Xaa 15  Xaa 16  Trp; 
 where Xaa 1 , Xaa 2 , Xaa 3 , Xaa 4 , Xaa 5 , Xaa 6 , Xaa 7 , Xaa 8 , Xaa 9 , Xaa 10 , Xaa 11  Xaa 12 , Xaa 13 . Xaa 14 , Xaa 15 , and Xaa 16  are selected independently from a natural accurring amino acid, provided that Xaa 1  and Xaa 7  are not Cys. 
 
     
     
         2 . An isolated polypeptide capable of catalysing the conversion of a α-hydroxyglycine to an α-amide, which polypeptide is selected from the group consisting of:
 (a) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 2-306 of SEQ ID No. 1; 
 (b) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 3-336 of SEQ ID No. 2; 
 (c) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 3-305 of SEQ ID No. 3; 
 (d) a polypeptide comprising an amino acid sequence having at least 80% identity to amino acids 3-279 of SEQ ID No. 4; and 
 (f) a polypeptide having peptidyl-α-hydroxyglycine alpha-amidating lyase activity characterised in that it has an amino acid sequence comprising no cysteine residues, or at most 1 or at most 2 cysteine residues. 
 
     
     
         3 . An isolated polypeptide having peptidyl-α-hydroxyglycine alpha-amidating lyase activity characterised in that it can be expressed as a soluble protein in  E. coli.    
     
     
         4 . The polypeptide of  claim 1 , comprising 0, 1 or 2 cysteine residues. 
     
     
         5 . The polypeptide of  claim 1 , which polypeptide is derived from a prokaryotic organism. 
     
     
         6 . The polypeptide of  claim 1  capable of catalysing the conversion of a α-hydroxyglycine to an α-amide, wherein said α-hydroxyglycine is a C-terminal-α-hydroxyglycine of formula R-Gly(OH), where R is a peptide and Gly(OH) is an α-hydroxyglycine residue linked to the C-terminus of said peptide, and
 wherein said α-amide is of formula R—NH 2 . 
 
     
     
         7 . A method for producing an enzyme with peptidyl-α-hydroxyglycine alpha-amidating lyase (PAL) activity comprising the steps of:
 (i) cultivating a recombinant expression host cell of non-mammalian origin, which comprises a nucleic acid construct comprising a nucleotide sequence encoding the enzyme, under conditions suitable for the expression of the enzyme; and 
 (ii) recovering the enzyme from
 (a) the supernatant after cell disruption and centrifugation and/or 
 (b) the growth media 
 
 wherein the host cell is a of non-mammalian origin, e.g. an  Escherichia coli  strain, and wherein said enzyme is soluble when recovered in step (ii). 
 
     
     
         8 . The method of  claim 7 , wherein the enzyme is in a catalytically active form when recovered in step (ii) meaning that the enzyme does not require a step of refolding for obtaining catalytic activity. 
     
     
         9 . An isolated polypeptide which is an enzyme with peptidyl-α-hydrozyglycine alpha-amidating lyase activity obtainable by the method of  claim 7  or  8 , wherein said enzyme is capable of catalysing the reaction:
   R-Gly(OH)→R—NH 2 ,
 
 
       where R is a peptide and Gly(OH) is an α-hydroxyglycine residue linked to the C-terminus of said peptide. 
     
     
         10 . A method for producing an enzyme having peptidyl-α-hydroxyglycine alpha-amidating lyase activity comprising maintaining a recombinant expression host cell comprising a recombinant nucleic acid comprising a nucleotide sequence encoding the polypeptide of  claim 1  under conditions suitable for the production of said enzyme. 
     
     
         11 . 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 of  claim 1  under conditions suitable for enzymatic activity, 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. 
 
     
     
         12 . A pharmaceutical composition comprising the recovered C-terminally α-amidated peptide of  claim 11 . 
     
     
         13 . The method of  claim 10 , wherein said 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.