Puromycine hydrolase (and also pQQM from Pseudomonas, C. Elegans dp4). Some Streptomyces strains produce Puromycin (PM classified into a family of nucleoside antibiotics) and express a PM-inactivating enzyme as a self-resistance determinant which is homologous to Peptidase S9
Puromycin-hydrolizing peptidases have been described as members of the prolyl oligopeptidase peptidase family. These enzymes are present across all domains of life but still little is known of the homologs found in the pathogenic bacterium Mycobacterium tuberculosis. The crystal structure of a M. tuberculosis puromycin hydrolase peptidase has been determined at 3 Angstrom resolution, revealing a conserved prolyl oligopeptidase fold, defined by alpha/beta-hydrolase and beta-propeller domains with two distinctive loops that occlude access of large substrates to the active site. The enzyme displayed amino peptidase activity with a substrate specificity preference for hydrophobic residues in the decreasing order of phenylalanine, leucine, alanine and proline. The enzyme's active site is lined by residues Glu564 for the coordination of the substrates amino terminal moiety and His561, Val608, Tyr78, Trp306, Phe563 and Ty567 for the accommodation of hydrophobic substrates. The availability of a crystal structure for puromycin hydrolase of M. tuberculosis shall facilitate the development of inhibitors with therapeutic applications. This article is protected by copyright. All rights reserved.
Puromycin (PM) is classified into a family of nucleoside antibiotics together with blasticidin S (BS). PM-producing Streptomyces alboniger is known to express a PM-inactivating enzyme as a self-resistance determinant, which catalyzes the acetylation of PM. We have shown that, although BS-producing Streptomyces morookaensis also produces a PM-inactivating enzyme, it catalyzes the hydrolysis of an amide linkage between the aminonucleoside and O-methyl-L-tyrosine moiety of PM. In the present study, we cloned and characterized a gene encoding PM hydrolase (PMH) from BS-producing S. morookaensis JCM4673. The nucleotide sequence analysis suggests that an open reading frame consisting of 1986 bp is a gene for PMH and encodes a protein consisting of 662 amino acids with a calculated molecular mass of 71,260 Da. The molecular mass of the recombinant PMH, which was produced using an Escherichia coli host-vector system, was the same as that of PMH purified from the JCM4673 strain. Our biochemical study of the recombinant PMH confirmed that the enzyme is an aminopeptidase with broad substrate specificity. The putative primary structure of PMH contains a Gly-X-Ser-X-Gly motif, which is commonly observed among serine proteases. In addition, the amino acid sequence of PMH displays a high similarity to that of the Streptomyces acyl-peptide hydrolase (ACPH), which is a member of the prolyl oligopeptidase (POP) family of serine proteases. Furthermore, the catalytic triad (Ser-Asp-His), which is observed in the POP family, is also present in the primary structure of PMH. These results suggest that PMH is an aminopeptidase classified into the POP family.
