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

摘要

GSP41 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 MgCl_2 and CaCl_2. Although Mg~(2+)- and Ca~(2+)-activated CSP41 cleave at identical sites in the single-stranded regions of a stem-loop-containing substrate, Mg~(2+)-activated CSP41 was also able to cleave within the double-stranded region of the stem-loop. Mixed metal experiments with Mg~(2+)- and Ca~(2+) suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Ma~(2+), Co~(2+) and Zn~(2+) compete with Mg~(2+) 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 metalbinding pocket. The activation constant for Mg~(2+) (K_(A,Mg) = 2.1 ± 0.4 mM) is of the same order of magnitude as the stromal Mg~(2+) concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg~(2+) concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.