Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily.
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+ (K(A,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.
Bollenbach
Thomas J
TJ
Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, NY 14853, USA. tjb26@cornell.edu
Stern
David B
DB
D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D002413:Cations, Divalent / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology*; D003573:Cytochrome b Group / Q000235:genetics; D045346:Cytochrome b6f Complex; D004722:Endoribonucleases / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000145:classification; D018749:RNA, Plant / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D002413:Cations, Divalent; D003573:Cytochrome b Group; D018749:RNA, Plant; C084072:petD protein, cytochrome b(6)f complex; D045346:Cytochrome b6f Complex; D010088:Oxidoreductases; D004722:Endoribonucleases; C106104:endoribonuclease CSP41; D008274:Magnesium; D002118:Calcium