Literature summary for 2.1.1.300 extracted from

Torres, M.A.; Hoffarth, E.; Eugenio, L.; Savtchouk, J.; Chen, X.; Morris, J.S.; Facchini, P.J.; Ng, K.K.
Structural and functional studies of pavine N-methyltransferase from Thalictrum flavum reveal novel insights into substrate recognition and catalytic mechanism (2016), J. Biol. Chem., 291, 23403-23415 .

Search for more literature for 2.1.1.300

Cloned(Commentary)

Cloned (Comment)	Organism
DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of His6-tagged wild-type and mutant enzymes and of selenomethionine-substituted enzyme in Escherichia coli	Thalictrum flavum subsp. glaucum

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant native and selenomethionine-substituted wild-type and mutant H206A enzymes in complex with S-adenosyl-L-methionine, of wild-type apoenzyme, and of wild-type enzyme in complex with S-adenosyl-L-homocysteine (SAH) or with SAH and tetrahydropapaverine, hanging drop vapour diffusion method, mixing of 0.0015 ml of protein solution with 0.0015 ml of reservoir solution containing 340 g/l pentaerythritol ethoxylate (15:4 EO/OH), 50 mM bis-Tris-HCl, pH 6.0, 70 mM ammonium sulfate, and 160 g/l glycerol, and equilibration against 0.45 ml of reservoir solution at 22°C, 1-3 weeks, for the complex crystals, the enzyme is incubated with the ligands (0.75 mM THP oand/or 1 mM SAM/SAH) before crystallization, method optimization, X-ray diffraction structure determination and analysis at 1.6-2.3 A resolution	Thalictrum flavum subsp. glaucum

Crystallization (Comment)

Organism

purified recombinant native and selenomethionine-substituted wild-type and mutant H206A enzymes in complex with S-adenosyl-L-methionine, of wild-type apoenzyme, and of wild-type enzyme in complex with S-adenosyl-L-homocysteine (SAH) or with SAH and tetrahydropapaverine, hanging drop vapour diffusion method, mixing of 0.0015 ml of protein solution with 0.0015 ml of reservoir solution containing 340 g/l pentaerythritol ethoxylate (15:4 EO/OH), 50 mM bis-Tris-HCl, pH 6.0, 70 mM ammonium sulfate, and 160 g/l glycerol, and equilibration against 0.45 ml of reservoir solution at 22°C, 1-3 weeks, for the complex crystals, the enzyme is incubated with the ligands (0.75 mM THP oand/or 1 mM SAM/SAH) before crystallization, method optimization, X-ray diffraction structure determination and analysis at 1.6-2.3 A resolution

Thalictrum flavum subsp. glaucum

Protein Variants

Protein Variants	Comment	Organism
E205A	site-directed mutagenesis, replacement of the residue with Ala results in a small decrease in protein stability as observed through a slight reduction in melting temperature, and to a large decrease in activity for three different substrates, especially the best substrate identified so far, (S)-reticuline	Thalictrum flavum subsp. glaucum
E205A/H206A	site-directed mutagenesis, replacement of Glu205 and His206 with Ala leads to large decreases in activity for three different substrates to about 10% of wild-type activity at pH 7.5	Thalictrum flavum subsp. glaucum
E80A	site-directed mutagenesis, the mutant shows 2fold increased activity compared to wild-type with substrate (S)-reticuline	Thalictrum flavum subsp. glaucum
H206A	site-directed mutagenesis, substrate binding structures compared to the wild-type enzyme, overview. Replacement of the residue with Ala results in a small decrease in protein stability as observed through a slight reduction in melting temperature, and to a large decrease in activity for three different substrates, especially the best substrate identified so far, (S)-reticuline	Thalictrum flavum subsp. glaucum
Y79A	site-directed mutagenesis, inactive mutant	Thalictrum flavum subsp. glaucum

Inhibitors

Inhibitors	Comment	Organism	Structure
(S)-tetrahydropapaverine	substrate inhibition at higher concentrations, enzyme complex structure with S-adenosyl-L-homocysteine	Thalictrum flavum subsp. glaucum
additional information	no substrate inhibition by (S)-reticuline	Thalictrum flavum subsp. glaucum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	steady-state kinetics	Thalictrum flavum subsp. glaucum
0.016	-	(S)-tetrahydropapaverine	pH 7.5, 30°C, recombinant enzyme	Thalictrum flavum subsp. glaucum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
S-adenosyl-L-methionine + (+/-)-N-methylpavine	Thalictrum flavum subsp. glaucum	-	S-adenosyl-L-homocysteine + (+/-)-N,N-dimethylpavine	-	?
S-adenosyl-L-methionine + (+/-)-pavine	Thalictrum flavum subsp. glaucum	-	S-adenosyl-L-homocysteine + (+/-)-N-methylpavine	-	?
S-adenosyl-L-methionine + (S)-tetrahydropapaverine	Thalictrum flavum subsp. glaucum	-	S-adenosyl-L-homocysteine + (S)-laudanosine	-	-

Organism

Organism	UniProt	Comment	Textmining
Thalictrum flavum subsp. glaucum	C3SBW0	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme from Escherichia coli by nickel affinity chromatography, dialysis, anion exchange chromatography, and ultrafiltration	Thalictrum flavum subsp. glaucum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	analysis of substrate specificity by mass spectrometry, overview. Enzyme PavNMT exhibits a preference for (+)-pavine and the 1-benzylisoquinoline (S)-reticuline, but also accepts the protoberberines scoulerine and stylopine and, to a lesser extent, tetrahydropapaverine (THP). No activity with (R)-reticuline and papaverine, as well as cryptopine, glaucine, codeine, canadaline, noscapine, and berbamine. Structural determinants of substrate binding are derived from crystal structure analysis	Thalictrum flavum subsp. glaucum	?	-	-
S-adenosyl-L-methionine + (+/-)-N-methylpavine	-	Thalictrum flavum subsp. glaucum	S-adenosyl-L-homocysteine + (+/-)-N,N-dimethylpavine	-	?
S-adenosyl-L-methionine + (+/-)-pavine	-	Thalictrum flavum subsp. glaucum	S-adenosyl-L-homocysteine + (+/-)-N-methylpavine	-	?
S-adenosyl-L-methionine + (R,S)-scoulerine	-	Thalictrum flavum subsp. glaucum	S-adenosyl-L-homocysteine + N-methylscoulerine	-	?
S-adenosyl-L-methionine + (R,S)-stylopine	-	Thalictrum flavum subsp. glaucum	S-adenosyl-L-homocysteine + N-methylstylopine	-	?
S-adenosyl-L-methionine + (S)-reticuline	best substrate. (R)-reticuline is not accepted as a substrate, revealing a strong stereoselectivity for (S)-reticuline	Thalictrum flavum subsp. glaucum	S-adenosyl-L-homocysteine + (S)-tetrahydropapaverine	-	?
S-adenosyl-L-methionine + (S)-tetrahydropapaverine	-	Thalictrum flavum subsp. glaucum	S-adenosyl-L-homocysteine + (S)-laudanosine	-	-
S-adenosyl-L-methionine + (S)-tetrahydropapaverine	i.e. THP, low activity, a racemic mixture of THP is converted by PavNMT to laudanosine. The benzyl group of (S)-THP is most deeply buried in the substrate-binding pocket, whereas the isoquinoline moiety lies in a more exposed position where the plane of the six-carbon ring is stacked in a parallel arrangement against the benzyl group of a molecule of (R)-THP. The molecule of (R)-THP appears to be bound more loosely, with fewer direct contacts with the protein than seen for the molecule of (S)-THP. The loop formed by residues 75-91 covers both THP molecules, and the structure of this loop appears to be dependent on the binding of both THP molecules, because it is disordered when THP is not bound	Thalictrum flavum subsp. glaucum	S-adenosyl-L-homocysteine + (S)-laudanosine	-	-

Subunits

Subunits	Comment	Organism
dimer	PavNMT forms a dimer with 2fold rotational symmetry in the asymmetric unit that is not seen in homologous NMTs	Thalictrum flavum subsp. glaucum
More	the overall structure of PavNMT reveals the presence of an alpha/beta SAM-binding domain with a canonical Rossmann fold that is shared among most SAM-dependent methyltransferases. In addition to this domain, a primarily alpha-helical BIA-binding domain forms the majority of the putative binding site for the methyl group acceptor	Thalictrum flavum subsp. glaucum

Synonyms

Synonyms	Comment	Organism
pavine NMT	-	Thalictrum flavum subsp. glaucum
PfPavNMT	-	Thalictrum flavum subsp. glaucum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Thalictrum flavum subsp. glaucum

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.00079	-	(S)-tetrahydropapaverine	pH 7.5, 30°C, recombinant enzyme	Thalictrum flavum subsp. glaucum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Thalictrum flavum subsp. glaucum

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	SAM, enzyme binding structure, overview	Thalictrum flavum subsp. glaucum

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.12	-	(S)-tetrahydropapaverine	pH 7.5, 30°C, recombinant enzyme	Thalictrum flavum subsp. glaucum

General Information

General Information	Comment	Organism
metabolism	the enzyme is involved in benzylisoquinoline alkaloids biosynthesis	Thalictrum flavum subsp. glaucum
additional information	substrate recognition and catalytic mechanism of pavine N-methyltransferase from Thalictrum flavum, highly conserved residues at the active site, overview. The loop formed by residues 75-91 covers both THP molecules, and the structure of this loop appears to be dependent on the binding of both THP molecules, because it is disordered when THP is not bound	Thalictrum flavum subsp. glaucum