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Molecular cloning and characterization of gene encoding novel puromycin-inactivating enzyme from blasticidin
Motohiro Nishimura1, Kayo Ikeda, Masanori Sugiyama
1Department of Chemical and Biological Engineering, Ube National College of Technology, 2-14-1 Tokiwadai, Ube, Yamaguchi 755-8555, Japan. nisimura@ube-k.ac.jp
Abstract:
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.
Insights
Researchers cloned and characterized puromycin hydrolase (PMH) from Streptomyces morookaensis. This enzyme, an aminopeptidase, inactivates puromycin by hydrolysis and belongs to the prolyl oligopeptidase family of serine proteases.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Puromycin (PM) is a nucleoside antibiotic.
- PM-inactivating enzymes confer self-resistance in producing organisms.
- Streptomyces morookaensis produces a PM-inactivating enzyme that hydrolyzes PM.
Purpose of the Study:
- To clone and characterize the gene encoding puromycin hydrolase (PMH) from Streptomyces morookaensis.
- To elucidate the biochemical properties and classification of PMH.
Main Methods:
- Gene cloning and nucleotide sequencing of PMH from S. morookaensis.
- Recombinant PMH expression in Escherichia coli.
- Biochemical characterization of recombinant PMH.
- Sequence analysis and comparison with known proteases.
Main Results:
- The PMH gene consists of 1986 bp, encoding a 662-amino acid protein.
- Recombinant PMH exhibited hydrolytic activity against PM, confirming its function.
- Biochemical studies identified PMH as an aminopeptidase with broad substrate specificity.
- Sequence analysis revealed PMH belongs to the prolyl oligopeptidase (POP) family of serine proteases, containing conserved motifs and catalytic triad.
Conclusions:
- PMH from S. morookaensis is a novel aminopeptidase.
- PMH is classified within the prolyl oligopeptidase family of serine proteases.
- The characterization of PMH provides insights into antibiotic resistance mechanisms and enzyme evolution.
