Literature summary for 2.1.1.171 extracted from

Kumar, A.; Saigal, K.; Malhotra, K.; Sinha, K.M.; Taneja, B.
Structural and functional characterization of Rv2966c protein reveals an RsmD-like methyltransferase from Mycobacterium tuberculosis and the role of its N-terminal domain in target recognition (2011), J. Biol. Chem., 286, 19652-19661.

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Cloned(Commentary)

Cloned (Comment)	Organism
gene rsmD, complementation of rsmD-deleted Escherichia coli KL16DELTArsmD cells, expression of Rv2966c with His10-Smt3 fusion at the N-terminus from plasmid pMTase1 in Escherichia coli strain BL21 (DE3)	Mycobacterium tuberculosis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant Rv2966c, hanging drop vapour diffusion method, mixing of 0.004 ml of 17 mg/ml Rv2966c in 10 mM potassium phosphate buffer, pH 7.4, 5 mM EDTA, 10% v/v glycerol, 0.1% v/v 2-mercaptoethanol, 50 mM NaCl, 0.1 mM S-adenosyl-L-methionine, with 0.002 ml of reservoir solution containing 100 mM Tris-HCl, pH 7.4, 3.0 M potassium acetate at 24 °C, 2-3 days, X-ray diffraction structure determination and analysis at 1.9 A resolution, molecular replacement	Mycobacterium tuberculosis

Crystallization (Comment)

Organism

purified recombinant Rv2966c, hanging drop vapour diffusion method, mixing of 0.004 ml of 17 mg/ml Rv2966c in 10 mM potassium phosphate buffer, pH 7.4, 5 mM EDTA, 10% v/v glycerol, 0.1% v/v 2-mercaptoethanol, 50 mM NaCl, 0.1 mM S-adenosyl-L-methionine, with 0.002 ml of reservoir solution containing 100 mM Tris-HCl, pH 7.4, 3.0 M potassium acetate at 24 °C, 2-3 days, X-ray diffraction structure determination and analysis at 1.9 A resolution, molecular replacement

Mycobacterium tuberculosis

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	P95128	gene rv2966c	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant Rv2966c with His10-Smt3 fusion at the N-terminus from Escherichia coli strain BL21 (DE3) by nickel affinity and anion exchange chromatography	Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	the N-terminal domain of Rv2966c is important for binding not only RNA but also DNA substrates, binding of protein to DNA is much stronger than it is to RNA	Mycobacterium tuberculosis	?	-	?
S-adenosyl-L-methionine + guanine966 in 16S rRNA	recombinant Rv2966c can methylate G966 of 16S rRNA in vitro	Mycobacterium tuberculosis	S-adenosyl-L-homocysteine + N2-methylguanine966 in 16S rRNA	-	?
S-adenosyl-L-methionine + guanine966 in 30S rRNA	recombinant Rv2966c can use 30S ribosomes purified from rsmD-deleted Escherichia coli as substrate	Mycobacterium tuberculosis	S-adenosyl-L-homocysteine + N2-methylguanine966 in 30S rRNA	-	?

Subunits

Subunits	Comment	Organism
More	protein Rv2966c shows a two-domain structure with a short hairpin domain at the N-terminus and a C-terminal domain with the S-adenosylmethionine-methyltransferase-fold. The N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition. In contrast to the variable substrate binding domain, the C-terminal domain is a highly conserved structure consisting of the classical S-adenosyl-L-methionine-methyltransferase-fold found in several S-adenosyl-L-methionine-dependent methyltransferases. It consists of a central eight-stranded beta-sheet flanked by alpha-helices on both sides, the first five strands of the beta-sheet are parallel to each other and encompass the S-adenosyl-L-methionine binding site toward their C terminus end, whereas the remaining strands of the beta-sheet are antiparallel. The N-terminus of Rv2966c is the target recognition region that helps in binding to nucleic acids	Mycobacterium tuberculosis

Synonyms

Synonyms	Comment	Organism
m2G966 methyltransferase	-	Mycobacterium tuberculosis
RsmD-like methyltransferase	-	Mycobacterium tuberculosis
Rv2966c	-	Mycobacterium tuberculosis
Rv2966c protein	-	Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Mycobacterium tuberculosis

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	binding structure, overview	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
evolution	structural similarities with other methyltransferases are limited to the core S-adenosyl-L-methionine domain but not the target recognition domain	Mycobacterium tuberculosis
malfunction	truncated constructs of Rv2966c lacking the N-terminal 18- or 23-amino acid residues and the mutant protein 3RM do not show any significant binding to either DNA or RNA nor any m2G966 methyltransferase activity	Mycobacterium tuberculosis
additional information	the N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition	Mycobacterium tuberculosis