PMID: 9171407Jun 1, 1997Paper

Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

Journal of Bacteriology
V J HarwoodH J Schreier

Abstract

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM). At 100 degrees C, the enzyme displayed some tolera...Continue Reading

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Citations

Dec 14, 1999·Protein Science : a Publication of the Protein Society·T C ChengS I Chan
May 23, 2006·FEBS Letters·Tünde JuhászLászló Polgár
Aug 5, 2015·Applied Microbiology and Biotechnology·Nicole MikaMartin Rühl
Jun 23, 2010·Archives of Insect Biochemistry and Physiology·Charles DarkohBeatrice Clack
Nov 19, 2010·The Journal of Biological Chemistry·Veronika HarmatLászló Polgár
Mar 30, 2001·The Journal of Biological Chemistry·M N HarrisV J Harwood
Jul 25, 2015·Nature Communications·Poonam SrivastavaJared C Lewis
Dec 1, 2018·Emerging Topics in Life Sciences·Julie A Maupin-Furlow
May 21, 2013·Journal of Industrial Microbiology & Biotechnology·Chao KangYan Xu
Jul 22, 2020·Chemical Communications : Chem Comm·Woo Jae JeongWoon Ju Song
Apr 11, 2021·Applied Microbiology and Biotechnology·Viia Kõiv, Tanel Tenson
Oct 11, 2003·Biotechnology Advances·J Eichler
Jan 2, 2007·Neuropeptides·J A García-HorsmanJ I Venäläinen
Feb 21, 2019·Biochemistry·Ken Ellis-GuardiolaJared C Lewis
Nov 26, 2008·Pharmacology & Therapeutics·Uros V DjekicNathaniel M Weathington

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