Tetrahydroprotoberbine N-methyltransferase is a protein that overall binding pocket adopts an “L-shape” which is complementary to a conformation that only the (S)-cis configuration of N-methylstylopine can readily adopt. The other product of the reaction, S-adenosyl-L-homocysteine (SAH), is found proximal to the CP within the cofactor binding site.
Function(s) and Biological Relevance
TNMT involved in the biosynthesis of protopine and benzophenanthridine alkaloids. Tetrahydroprotoberberine N-methyltransferase catalyzes the conversion of the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. No activity with dimethoxytetrahydroisoquinoline, methylisoquinolinediol, norlaudanosoline, (R,S)-tetrahydroxyberbine, (S)-scoulerine or (R,S)-pavine as substrates.
The substrate specificity of TNMT enzymes appears to arise from the arrangement of subgroup-specific stereospecific recognition elements relative to catalytic elements that are more widely-conserved. This research will provide descriptive roles that TNMT plays such as pathway leading to the formation of different substrates including Protoberberine.
Broader Implications
N-methylation is a recurring feature in the biosynthesis of many plant specialized metabolites, including alkaloids. A crucial step in the conserved central pathway that provides intermediates for the biosynthesis of benzylisoquinoline alkaloids (BIAs). Prominent compounds include the narcotic analgesic morphine, the cough suppressant codeine, the muscle relaxants papaverine and (+)‐tubocurarine, the anti‐microbial agent sanguinarine, and the cholesterol‐lowering drug berberine. In this study, the stereoselectivity of the yellow horned poppy’s enzyme controls what substrates can interact, the products you will get, and how much medicinal compound you can extract from the plant. In TMNT, three amino acid residues in the alpha14-helix form one side of the binding pocket defining the BP region. The binding pocket consists of His-328(green), Ile-329(purple), and Phe-332(orange). The H328 mutation decreases in activity with stylopine and scoulerine producing a 5- and 2-fold while the activity with THP increases 2-fold.
Structural highlights and structure-function relationships
GfTNMT was co-crystallized with the cofactor S-adenosyl-L-methionine (dmin = 1.6 A), product S-adenosyl-L-homocysteine (dmin = 1.8 A), or in complex with S-adenosyl-L-homocysteine and (S)-cis-N-methylstylopine (dmin = 1.8 A), The of this protein contains a pattern of hydrogen bonds between atoms in the peptide bond. These structures reveal for the first time how a mostly L-shaped substrate recognition pocket selects for the (S)-cis configuration of the two central six-membered rings in protoberberine BIA compounds. There are of SAM that form a small catalytic pocket and surrounds the amino group and methyl donor of SAM. The substrate specificity of TNMT enzymes appears to arise from the of subgroup-specific stereospecific recognition elements relative to catalytic elements that are more widely conserved among all BIA NMTs. The basic view of the entire protein allows readers to visualize the different elements show in different colors. The elements shown are carbons(grey), nitrogen(blue), and oxygen(red). It's consists of His-208, Glu-204, and Glu-207. The mutagenesis studies confirm and functionally define the roles of several highly conserved residues within and near the GfTNMT protein consists of amino acids Valine-188(yellow), Aspartic Acid-187(blue), and Alanine-186(green), with purple being the rest of the ligand. The SAM effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. The binding mode of protoberberine compounds to GfTNMT appears to be similar to coclaurine NMT, with the isoquinoline rings buried deepest in the binding pocket. This binding mode differs from that of pavine NMT, in which the benzyl ring is bound more deeply than the isoquinoline rings. The insights into and catalysis provided here form a sound basis for the rational engineering of for chemoenzymatic synthesis and metabolic engineering.
Energy Transformation
Full-length cDNAs for the three SOMT candidates were cloned into the pRSETA expression vectorwithan N-terminal His6 tag translational fusion. Recombinant SOMT1, SOMT2, and SOMT3 were purified from total protein extract using a cobalt-affinity resin. The recombinant enzymes displayed molecular mass values of approximately 43, 40, and 39 kD as determined by SDS-PAGE, which were marginally higher than the predicted values owing to the N-terminal peptide fusion. The enzyme has a pH optimum of 8.9, a temperature optimum at 40° and a Mr of about 78 000 ± 10%. The Km for (S)-canadine was determined to be 6.4,μM, for (S)-stylopine 3.1 μM and for SAM 12,μM. The enzyme is inhibited by S-adenosyl-l-homocysteine (SAH with a Ki of 24 μM.
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