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J. Bacteriol. doi:10.1128/JB.00396-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The N-terminal propeptide of Vibrio vulnificus extracellular metalloprotease is both an inhibitor and substrate for the enzyme

Research Center for Proteineous Materials, Chosun University, Gwangju 501-759, Republic of Korea; Department of Biotechnology and BK21 Research Team for Protein Activity Control, College of Natural Sciences, Chosun University, Gwangju 501-759, Republic of Korea

^* To whom correspondence should be addressed. Email: jsplee{at}mail.chosun.ac.kr.

	Abstract

Vibrio vulnificus, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions; a signal peptide, an N-terminal propeptide, a C-terminal propeptide, and the mature protease. Two forms of vEP; vEP-45 which contains the mature protease plus the C-terminal propeptide, and vEP-34 which contains only the mature protease were expressed in Escherichia coli and purified. Both vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The N-terminal propeptide (nPP) of vEP was expressed as a his tag fusion protein and its effect on vEP activity was investigated. nPP inhibited the activity of both vEP-45 and vEP-34, but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under condition of low enzyme and propeptide concentrations, and with prolonged incubation, a process that resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, the inhibition of vEP by nPP at high temperature was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the N-terminal propeptide of vEP could bind to vEP and specially inhibit its activity, resulting in the degradation of the propeptide.