Overexpression and characterization of carboxyl-terminal processing protease for precursor D1 protein. Regulation of enzyme-substrate interaction by molecular environments

Yumiko Yamamoto, Noritoshi Inagaki, Kimiyuki Satoh

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)7518-7525
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number10
DOIs
Publication statusPublished - Mar 9 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Overexpression and characterization of carboxyl-terminal processing protease for precursor D1 protein. Regulation of enzyme-substrate interaction by molecular environments'. Together they form a unique fingerprint.

  • Cite this