Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily

• Published in: Nucleic acids research 2003-08, Vol.31 (15), p.4317-4325
• Main Authors: Bollenbach, T.J, Stern, D.B
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Calcium - metabolism; Calcium - pharmacology; Catalysis; Cations, Divalent - pharmacology; chloroplasts; Chloroplasts - enzymology; Cytochrome b Group - genetics; Cytochrome b6f Complex; endoribonucleases; Endoribonucleases - chemistry; Endoribonucleases - classification; Endoribonucleases - metabolism; enzyme activity; Magnesium - metabolism; Magnesium - pharmacology; metal ions; molecular conformation; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidoreductases - classification; RNA; RNA cleavage; RNA, Plant - chemistry; RNA, Plant - metabolism; Sequence Alignment; stem loop structure; Substrate Specificity
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkg640

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5′ to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold‐containing proteins from the SDR superfamily, including several RNA‐binding proteins and endonucleases. CSP41 is active only in the presence of MgCl2 and CaCl2. Although Mg2+‐ and Ca2+‐activated CSP41 cleave at identical sites in the single‐stranded regions of a stem–loop‐containing substrate, Mg2+‐activated CSP41 was also able to cleave within the double‐stranded region of the stem–loop. Mixed metal experiments with Mg2+ and Ca2+ suggest that CSP41 contains a single divalent metal‐binding site which is non‐selective, since Mn2+, Co2+ and Zn2+ compete with Mg2+ for binding, although there is no activity in their presence. Using site‐directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal‐binding pocket. The activation constant for Mg2+ (KA,Mg = 2.1 ± 0.4 mM) is of the same order of magnitude as the stromal Mg2+ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg2+ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.