Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.3 (RNase III)
 1,015 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs. RNase III is a homodimer and requires Mg(2+) to hydrolyze phosphodiesters. The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding [Li and Nicholson (1996) EMBO J. 15, 1421-1433]. To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid. The mutant proteins were purified as (His)(6)-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking. The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate. The Glu117Asp mutant also is defective in cleavage while able to bind substrate. However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency approximately 15 000-fold lower than that of RNase III. The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn(2+) for Mg(2+), with the catalytic efficiency of the Glu117Asp-Mn(2+) holoenzyme approximately 400-fold lower than that of the RNase III-Mn(2+) holoenzyme. For RNase III, a Mn(2+) concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory. In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn(2+). Finally, high concentrations of Mg(2+) do not inhibit RNase III nor relieve the Mn(2+)-dependent inhibition. In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III. One site binds either Mg(2+) or Mn(2+) and supports catalysis, while the other site is specific for Mn(2+) and confers inhibition. Glu117 is important for the function of both sites. The implications of these findings on the RNase III catalytic mechanism are discussed.
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PMID:Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant. 1130 28

The double strand-specific endoRNase RNase III globally regulates the production of antibiotics by Streptomyces coelicolor. We have undertaken studies to determine whether the endoRNase activity of S. coelicolor RNase III or its RNA binding activity is responsible for its regulatory function. We show that an rnc null mutant of S. coelicolor M145 does not produce actinorhodin or undecylprodigiosin. Restoring a wild-type copy of rnc to that mutant also restored antibiotic production. We constructed an rnc point mutant, D70A, in which an aspartic acid residue which is essential for the catalytic activity of RNase III was changed to alanine. The D70A mutation abolished the catalytic activity of the protein but not its ability to bind to RNA substrates. Introduction of a copy of the D70A gene into the rnc null mutant did not restore antibiotic production. This result suggests that the endoRNase activity of RNase III is required for the regulation of antibiotic production in S. coelicolor. We also reconstructed the C120 point mutation that was originally described in 1992. Although that mutation diminished antibiotic production by S. coelicolor, we confirm here that the C120 protein retains some RNase III activity.
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PMID:The endonuclease activity of RNase III is required for the regulation of antibiotic production by Streptomyces coelicolor. 1895 7