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Originally published In Press as doi:10.1074/jbc.M008877200 on November 30, 2000

J. Biol. Chem., Vol. 276, Issue 10, 7518-7525, March 9, 2001
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Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein
REGULATION OF ENZYME-SUBSTRATE INTERACTION BY MOLECULAR ENVIRONMENTS*

Yumiko YamamotoDagger , Noritoshi Inagaki§, and Kimiyuki SatohDagger ||

From the Dagger  Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan and the § National Institute for Basic Biology, Okazaki 444-8585, Japan

CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions.

* This work was supported by Grant-in-aid for Scientific Research (B) No. 09440268 from the Ministry of Education, Science, Sports, and Culture and by a grant from the Okayama Foundation for Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Lab. of Photosynthesis, National Inst. of Agrobiological Resources, Tsukuba 305-8602, Japan.

|| To whom correspondence should be addressed. Tel.: 81-86-251-7862; Fax: 81-86-251-7877; E-mail: kimiyuki@cc.okayama-u.ac.jp.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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