Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary 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 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.300 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)

EC Number Crystallization (Comment) Organism
2.1.1.300 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

EC Number Protein Variants Comment Organism
2.1.1.300 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
2.1.1.300 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
2.1.1.300 E80A site-directed mutagenesis, the mutant shows 2fold increased activity compared to wild-type with substrate (S)-reticuline Thalictrum flavum subsp. glaucum
2.1.1.300 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
2.1.1.300 Y79A site-directed mutagenesis, inactive mutant Thalictrum flavum subsp. glaucum

Inhibitors

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.300 additional information
-
 additional information steady-state kinetics Thalictrum flavum subsp. glaucum
2.1.1.300 0.016
-
 (S)-tetrahydropapaverine pH 7.5, 30°C, recombinant enzyme Thalictrum flavum subsp. glaucum

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.300 Thalictrum flavum subsp. glaucum C3SBW0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.300 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.300 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 ?
-
-
2.1.1.300 S-adenosyl-L-methionine + (+/-)-N-methylpavine
-
 Thalictrum flavum subsp. glaucum S-adenosyl-L-homocysteine + (+/-)-N,N-dimethylpavine
-
 ?
2.1.1.300 S-adenosyl-L-methionine + (+/-)-pavine
-
 Thalictrum flavum subsp. glaucum S-adenosyl-L-homocysteine + (+/-)-N-methylpavine
-
 ?
2.1.1.300 S-adenosyl-L-methionine + (R,S)-scoulerine
-
 Thalictrum flavum subsp. glaucum S-adenosyl-L-homocysteine + N-methylscoulerine
-
 ?
2.1.1.300 S-adenosyl-L-methionine + (R,S)-stylopine
-
 Thalictrum flavum subsp. glaucum S-adenosyl-L-homocysteine + N-methylstylopine
-
 ?
2.1.1.300 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
-
 ?
2.1.1.300 S-adenosyl-L-methionine + (S)-tetrahydropapaverine
-
 Thalictrum flavum subsp. glaucum S-adenosyl-L-homocysteine + (S)-laudanosine
-
-
2.1.1.300 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

EC Number Subunits Comment Organism
2.1.1.300 dimer PavNMT forms a dimer with 2fold rotational symmetry in the asymmetric unit that is not seen in homologous NMTs Thalictrum flavum subsp. glaucum
2.1.1.300 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

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.300 30
-
 assay at Thalictrum flavum subsp. glaucum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.1.1.300 0.00079
-
 (S)-tetrahydropapaverine pH 7.5, 30°C, recombinant enzyme Thalictrum flavum subsp. glaucum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.300 7.5
-
 assay at Thalictrum flavum subsp. glaucum

Cofactor

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

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.1.1.300 0.12
-
 (S)-tetrahydropapaverine pH 7.5, 30°C, recombinant enzyme Thalictrum flavum subsp. glaucum

General Information

EC Number General Information Comment Organism
2.1.1.300 metabolism the enzyme is involved in benzylisoquinoline alkaloids biosynthesis Thalictrum flavum subsp. glaucum
2.1.1.300 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