Dataset Information

Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor.

ABSTRACT: The cleavage of double-stranded (ds) RNA by ribonuclease III is a conserved early step in bacterial rRNA maturation. Studies on the mechanism of dsRNA cleavage by RNase III have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, neither the catalytic properties of extremophile RNases III nor the structures and reactivities of their cognate substrates have been described. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima was analyzed using purified recombinant enzyme. T. maritima (Tm) RNase III catalytic activity exhibits a broad optimal temperature range of approximately 40-70 degrees C, with significant activity at 95 degrees C. Tm-RNase III cleavage of substrate is optimally supported by Mg(2+) at >or=1 mM concentrations. Mn(2+), Co(2+), and Ni(2+) also support activity but with reduced efficiencies. The enzyme functions optimally at pH 8 and approximately 50-80 mM salt concentrations. Small RNA hairpins that incorporate the 16S and 23S pre-rRNA stem sequences are efficiently cleaved by Tm-RNase III at sites that are consistent with production in vivo of the immediate precursors to the mature rRNAs. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive recognition determinant for Escherichia coli (Ec) RNase III substrates. The dependence of reactivity on the pb sequence is similar to that observed with Ec-RNase III substrates. In fact, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity and is inhibited by antideterminant bp that also inhibit Ec-RNase III. These results indicate the conservation, across a broad phylogenetic distance, of positive and negative determinants of reactivity of bacterial RNase III substrates.

SUBMITTER: Nathania L

PROVIDER: S-EPMC3482404 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor.

Nathania Lilian L Nicholson Allen W AW

Biochemistry 20100801 33

The cleavage of double-stranded (ds) RNA by ribonuclease III is a conserved early step in bacterial rRNA maturation. Studies on the mechanism of dsRNA cleavage by RNase III have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, neither the catalytic properties of extremophile RNases III nor the structures and reactivities of their cognate substrates have been described. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima ...[more]

PMID: 20677811

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Project description:Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

Dataset Information

Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor.

Publications

Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor.

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