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Published on: January 13, 2017
Purification and characterization of a puromycin-hydrolyzing enzyme from blasticidin S-producing Streptomyces
M Nishimura1, H Matsuo, A Nakamura
1Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai, Yamaguchi. nisimura@ube-k.ac.jp
Abstract:
Blasticidin S-producing Streptomyces morookaensis JCM4673 produces an enzyme which inactivates puromycin (PM) by hydrolyzing an amide linkage between its aminonucleoside and O-methyl-L-tyrosine moieties [Nishimura et al. (1995) FEMS Microbiol. Lett. 132, 95-100]. In this study, we purified to homogeneity the enzyme from the cell-free extracts of S. morookaensis. The molecular weight of PM-hydrolyzing enzyme, estimated by SDS-PAGE and gel filtration, was 68 and 66 kDa, respectively, suggesting that this protein is monomeric. The PM-hydrolyzing activity was strongly inhibited by Zn2+, Fe2+, Cu2+, Hg2+, and N-bromosuccinimide, but was stimulated by DTT. The optimum pH and temperature for PM-hydrolyzing activity were 8.0 and 45 degrees C, respectively. Several L-aminoacyl-beta-naphthylamides were good substrates for the enzyme, suggesting that the PM-inactivating enzyme has an aminopeptidase activity. The N-terminal sequence of the first 14 amino acids (Val-Ser-Thr-Ala-Pro-Tyr-Gly-Ala-Trp-Gln-Ser-Pro-Ile-Asp) of the enzyme showed no significant homology with any published hydrolase sequences.
Insights
This study purified a puromycin (PM)-inactivating enzyme from Streptomyces morookaensis. The enzyme exhibits aminopeptidase activity and its N-terminal sequence shows no homology to known hydrolases.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Streptomyces morookaensis JCM4673 produces an enzyme that inactivates puromycin (PM).
- This inactivation occurs via hydrolysis of an amide linkage within puromycin.
Purpose of the Study:
- To purify the puromycin-inactivating enzyme from S. morookaensis.
- To characterize the biochemical properties and N-terminal sequence of the enzyme.
Main Methods:
- Enzyme purification from cell-free extracts.
- Molecular weight determination using SDS-PAGE and gel filtration.
- Enzyme activity assays under varying conditions and substrate specificities.
- N-terminal amino acid sequencing.
Main Results:
- The enzyme was purified to homogeneity.
- Estimated molecular weight suggests a monomeric protein (66-68 kDa).
- Optimal activity at pH 8.0 and 45°C; inhibited by heavy metals and N-bromosuccinimide; stimulated by DTT.
- Demonstrated aminopeptidase activity with L-aminoacyl-beta-naphthylamides.
- N-terminal sequence showed no homology to known hydrolases.
Conclusions:
- The purified enzyme inactivates puromycin through hydrolysis.
- The enzyme possesses aminopeptidase activity.
- Its unique N-terminal sequence suggests it may be a novel hydrolase.
