Molecular cloning and characterization of gene encoding novel puromycin-inactivating enzyme from blasticidin S-producing Streptomyces morookaensis

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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.

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Bacterial strains, plasmids, and cultivation media

BS-producing S. morookaensis JCM 4673 (= KCC S-0673) was obtained from the Japan Collection of Microorganisms (JCM) at the Institute of Physical and Chemical Research, Wako. The spores, formed on a YEME medium (1% glucose, 0.5% Polypepton, 0.3% yeast extract, 0.3% malt extract, and 0.04% MgCl2·6H2O, pH 7.0) supplemented with 1.5% agar, were suspended in 0.85% NaCl containing 20% glycerol and stored at −70°C until use. The Streptomyces spores were inoculated and cultured in a YEME medium

Cloning of gene for PMH

Streptomyces genes, in general, have a strong preference for utilizing codons that contain G or C in the third position (18). This bias was reflected in the design and synthesis of the mixed oligonucleotides corresponding to the N-terminal amino acid sequence (4A-P-Y-G-A-W-Q-S-P-I-14D) (9) of PMH from S. morookaensis JCM4673. The mixed probes, labeled with fluorescein, had a GCICCITACGGIGCITGGCAIWSICCI ATCGAC sequence (where W is A or T, I is deoxyinosine, and S is C or G). The probes were

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