Literature summary for 2.1.1.244 extracted from

Dong, C.; Mao, Y.; Tempel, W.; Qin, S.; Li, L.; Loppnau, P.; Huang, R.; Min, J.
Structural basis for substrate recognition by the human N-terminal methyltransferase 1 (2015), Genes Dev., 29, 2343-2348.

Search for more literature for 2.1.1.244

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes NTMT1 with a thrombin cleavage site at the N-terminus in Escherichia coli strain BL21(DE3) codon plus RIL	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
recombinant purified full-length wild-type NTMT1 in complex with S-adenosyl-L-homocysteine and a peptide derived from either human (SPKRIA) or mouse (PPKRIA) RCC1, or crystals of NTMT1 in complex with S-adenosyl-L-homocysteine and either the RPK or YPK substrate peptide, sitting drop vapor diffusion method, mixing of 0.001 ml of 37 mg/ml protein and ligand in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 mM TECP, with 0.001 ml of reservoir solution containing 23-28% PET 3350 and 14-18% Tacsimate, pH 6.0, X-ray diffraction structure determination and analysis, molecular replacement	Homo sapiens

Crystallization (Comment)

Organism

recombinant purified full-length wild-type NTMT1 in complex with S-adenosyl-L-homocysteine and a peptide derived from either human (SPKRIA) or mouse (PPKRIA) RCC1, or crystals of NTMT1 in complex with S-adenosyl-L-homocysteine and either the RPK or YPK substrate peptide, sitting drop vapor diffusion method, mixing of 0.001 ml of 37 mg/ml protein and ligand in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 mM TECP, with 0.001 ml of reservoir solution containing 23-28% PET 3350 and 14-18% Tacsimate, pH 6.0, X-ray diffraction structure determination and analysis, molecular replacement

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D180K	site-directed mutagenesis, inactive mutant	Homo sapiens
D180Y	site-directed mutagenesis, inactive mutant	Homo sapiens
H140K	site-directed mutagenesis, inactive mutant	Homo sapiens
N168K	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Homo sapiens
W136F	site-directed mutagenesis, almost inactive mutant	Homo sapiens
W136I	site-directed mutagenesis, inactive mutant	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
me3-RCC1-6	product inhibition	Homo sapiens
S-adenosyl-L-homocysteine	product inhibition	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.0003	-	N-terminal-PPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.0016	-	N-terminal-YPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.004	-	N-terminal-RPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.0054	-	N-terminal-LPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.0073	0.0079	N-terminal-SPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.126	-	N-terminal-WPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.2	-	N-terminal-SPKRIA-[RCC1]	above, mutant W136F, pH 7.5, 37°C	Homo sapiens
0.263	-	N-terminal-SPKRIA-[RCC1]	mutant N168K, pH 7.5, 37°C	Homo sapiens

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged wild-type and mutant enzymes NTMT1 from Escherichia coli strain BL21(DE3) codon plus RIL by nickel affinity and anion exchange chromatography followed by gel filtration	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3 S-adenosyl-L-methionine + N-terminal-LPKRIA-[RCC1]	-	Homo sapiens	3 S-adenosyl-L-homocysteine + N-terminal-trimethyl-LPKRIA-[RCC1]	-	?
3 S-adenosyl-L-methionine + N-terminal-PPKRIA-[RCC1]	best substrate	Homo sapiens	3 S-adenosyl-L-homocysteine + N-terminal-trimethyl-PPKRIA-[RCC1]	-	?
3 S-adenosyl-L-methionine + N-terminal-RPKRIA-[RCC1]	-	Homo sapiens	3 S-adenosyl-L-homocysteine + N-terminal-trimethyl-RPKRIA-[RCC1]	-	?
3 S-adenosyl-L-methionine + N-terminal-SPKRIA-[RCC1]	high activity	Homo sapiens	3 S-adenosyl-L-homocysteine + N-terminal-trimethyl-SPKRIA-[RCC1]	-	?
3 S-adenosyl-L-methionine + N-terminal-WPKRIA-[RCC1]	-	Homo sapiens	3 S-adenosyl-L-homocysteine + N-terminal-trimethyl-WPKRIA-[RCC1]	-	?
3 S-adenosyl-L-methionine + N-terminal-YPKRIA-[RCC1]	-	Homo sapiens	3 S-adenosyl-L-homocysteine + N-terminal-trimethyl-YPKRIA-[RCC1]	-	?
S-adenosyl-L-methionine + N-terminal peptide sequence of a protein	all known substrates of NTMT1 contain the N-terminal consensus sequence XPK (X = S/P/A/G), although NTMT1 can also methylate peptides with X being F, Y, C, M, K, R, N, Q, or H in vitro, substrate specificity of NTMT1, overview. Structural basis for the specific N-terminal methylation of a consensus motif, XPK, by NTMT1, overview. Hexapeptides composed of the first six residues of RCC1, i.e. regulator of chromosome condensation 1, are recognized by the enzyme. The first residue within the consensus sequence of the NTMT1 substrates is anchored through a hydrogen bond with the conserved Asn168 of NTMT1 in a spacious binding pocket, which exposes the substrate's reactive alpha-amino group to S-adenosyl-L-methionine in the complex structures, and this very N-terminal residue can tolerate most residue substitutions except the negatively charged residues D and E. Asp180 and His140 can act as bases to facilitate deprotonation of the target alpha-N-terminal amino group. Catalytic reaction proceeds probably involving a SN1 mechanism, overview	Homo sapiens	S-adenosyl-L-homocysteine + methylated N-terminal peptide sequence of a protein	S-adenosyl-L-homocysteine is bound to NTMT1 in an extended conformation. The carboxylate moiety of SAH forms a salt bridge interaction with the highly conserved Arg74, and the ribosyl group stacks with the indole ring of Trp20. In addition, the adenine moiety of SAH is flanked by the hydrophobic side chains of Ile92 and Val137 and interacts with the main chain amide group of Leu119 and the side chain of Gln120 through hydrogen bonding	?

Subunits

Subunits	Comment	Organism
More	the structure of enzyme NTMT1 includes a typical methyltransferase Rossmann fold that consists of a seven-strand beta sheet and five alpha helixes, of which two alpha helixes (alpha6 and alpha7) pack on one side of the beta sheet, and the other three alpha helixes (alpha3, alpha4, and alpha5) pack on the other side of the beta sheet. In addition to the highly conserved Rossmann fold, enzyme NTMT1contains two unique structural elements distinct from other methyltransferases: a beta hairpin inserted between strand beta5 and helix alpha7 and an N-terminal extension consisting of two alpha helixes (alpha1 and alpha2) and one 310 helix. These two unique structural elements are extensively involved in substrate binding, suggesting their potential contributions to substrate specificity	Homo sapiens

Synonyms

Synonyms	Comment	Organism
alpha-N-terminal methyltransferase 1	-	Homo sapiens
NTMT1	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.00067	-	N-terminal-YPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.0007	-	N-terminal-SPKRIA-[RCC1]	about, mutant W136F, pH 7.5, 37°C	Homo sapiens
0.0008	-	N-terminal-SPKRIA-[RCC1]	mutant N168K, pH 7.5, 37°C	Homo sapiens
0.0015	-	N-terminal-RPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.00167	-	N-terminal-WPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.0017	-	N-terminal-SPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.0018	-	N-terminal-PPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.0018	-	N-terminal-LPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Homo sapiens

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	-	Homo sapiens

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
0.0033	-	wild-type enzyme, pH 7.5, 37°C	Homo sapiens	S-adenosyl-L-homocysteine
0.025	-	above, wild-type enzyme, pH 7.5, 37°C	Homo sapiens	me3-RCC1-6

General Information

General Information	Comment	Organism
additional information	the enzyme contains two characteristic structural elements, a beta hairpin and an N-terminal extension, that contribute to its substrate specificity. Identification of key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for alpha-N-terminal methylation, NTMT1 prefers an XPK sequence motif, catalytic mechanism for alpha-N-terminal methylation and overall structure of the NTMT1 ternary complexes, verview	Homo sapiens
physiological function	alpha-N-terminal methylation seems to regulate protein stability via N-end rule pathways or mediate proteinprotein interactions. The enzyme also mediates protein-DNA interactions between chromatin and regulator of chromatin condensation	Homo sapiens

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.00033	-	N-terminal-SPKRIA-[RCC1]	above, mutant W136F, pH 7.5, 37°C	Homo sapiens
0.003	-	N-terminal-SPKRIA-[RCC1]	mutant N168K, pH 7.5, 37°C	Homo sapiens
0.013	-	N-terminal-WPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.233	-	N-terminal-SPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.33	-	N-terminal-LPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.375	-	N-terminal-RPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
0.419	-	N-terminal-YPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens
6	-	N-terminal-PPKRIA-[RCC1]	wild-type enzyme, pH 7.5, 37°C	Homo sapiens