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

Paper ID Volume ID Publish Year Pages File Format Full-Text
22243 43263 2006 7 PDF Available
Title
Molecular cloning and characterization of gene encoding novel puromycin-inactivating enzyme from blasticidin S-producing Streptomyces morookaensis
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.

Keywords
Streptomyces morookaensis; blasticidin S; puromycin; puromycin hydrolase; serine protease; catalytic triadACPH, acylpeptide hydrolase; BLM, bleomycin; BMH, bleomycin hydrolase; BS, blasticidin S; IPTG, isopropyl-β-D-thiogalactopyranoside; PM, puromycin; P
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Molecular cloning and characterization of gene encoding novel puromycin-inactivating enzyme from blasticidin S-producing Streptomyces morookaensis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 101, Issue 1, January 2006, Pages 63–69
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us