Structure and Biocatalytic Scope of Coclaurine <i>N</i>‐Methyltransferase

Bennett, Matthew R.; Thompson, Mark L.; Shepherd, Sarah A.; Dunstan, Mark S.; Herbert, Abigail J.; Smith, Duncan R. M.; Cronin, Victoria A.; Menon, Binuraj R. K.; Levy, Colin; Micklefield, Jason

doi:10.1002/ange.201805060

Cited by 6 publications

(4 citation statements)

References 22 publications

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“…The enzymatic activity was assayed using the LeuK0 (4) and LeuA (3) as the substrates, respectively. The S-adenosyl-L -homocysteine (SAH) and three new product peaks LeuK2 (6), LeuK3 (7) and LeuA0 (8) were only detected in the presence of the substrate, LcsG, and SAM (Fig. S10 and 2B).…”

Section: Lcsg Functions As a Sam-dependent Methyltransferasementioning

confidence: 99%

“…To elucidate the structures of compounds LeuK2 (6), LeuK3 (7) and LeuA0 (8), we turned to HRESI-MS-MS analysis. The m/z values of fragments of each leucinostatins were presented in Table S2.…”

Section: Characterizations Of the Products Of Lcsg-catalyzed Reactionmentioning

confidence: 99%

“…According to the molecular weights of LeuK2 (6) and LeuK3 (7), their molecular formulas should be C 63 H 113 N 11 O 14 and C 64 H 115 N 11 O 14 , respectively. From the MS-MS experiment, they also presented similar fragments as LeuK0 (Fig.…”

Section: Characterizations Of the Products Of Lcsg-catalyzed Reactionmentioning

confidence: 99%

“…[1][2][3] NMTs are also employed in the biosynthetic pathways of natural products. [4][5][6][7] The N-methylations catalyzed by NMTs contribute signi cantly to changing the property of natural products by modifying their structures and in uencing their bioactivities. 8 In the biosynthesis of nonribosomal peptides (NRPs), NMTs often integrate into the nonribosomal peptides synthase (NRPS) as domains, while seldom acting as a freestanding enzyme.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum

Li,

Jiao

et al. 2023

Preprint

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N-methyltransferase (NMT)-catalyzed methylations are rarely reported at nonribosomal peptides (NRPs) terminuses. Here, we discovered a fungal NMT LcsG for the iterative terminal N-methyl formation of a family of NRPs, leucinostatins. Gene deletion suggested LcsG is essential to the methylation of leucinostatins. In vitro assay and HRESI-MS-MS analysis proved the methylation sites were the NH2, NHCH3 and N(CH3)2 in the C-terminal unit of various leucinostatins. Based on the protein structure predicted by artificial intelligence (AI), molecular docking, and site-directed mutagenesis, we proposed the catalytic mechanism of the LcsG-catalyzed reaction was an N atom coordinated by two negatively charged residues (Asp368, Asp395 for LcsG) towards the subsequent SN2 methylation. These findings not only provide an approach for enriching the variety of natural bioactivity of NPRs but also deepen the insight into the catalytic mechanism of N-methylation of NRPs.

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Section: Lcsg Functions As a Sam-dependent Methyltransferasementioning

confidence: 99%

Section: Characterizations Of the Products Of Lcsg-catalyzed Reactionmentioning

confidence: 99%

Section: Characterizations Of the Products Of Lcsg-catalyzed Reactionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum

Li,

Jiao

et al. 2023

Preprint

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Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways

et al. 2020

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S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy‐SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site‐directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co‐factor, carboxy‐S‐adenosyl‐l‐ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1‐carboxyethyl group to the substrate.

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Multienzyme One‐Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids

et al. 2021

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The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S‐Adenosylmethionine (SAM)‐dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi‐enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non‐natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C‐1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule.

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Structure and Biocatalytic Scope of Coclaurine N‐Methyltransferase

Cited by 6 publications

References 22 publications

Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum

Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum

Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways

Multienzyme One‐Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids

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