Structural and Functional Studies of Pavine N-Methyltransferase from Thalictrum flavum Reveal Novel Insights into Substrate Recognition and Catalytic Mechanism

Torres, Megan; Hoffarth, Elesha R.; Eugenio, Luiz; Savtchouk, J.; Chen, Xue; Morris, Jeremy S.; Facchini, Peter J.; Ng, Kenneth

doi:10.1074/jbc.m116.747261

Cited by 29 publications

(45 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The CNMT structure (Figures 2 & S3) reveals a typical class I methyltransferase α/β Rossmann fold forming the AdoMet‐binding domain, which is capped by a predominantly alpha helical BIA substrate recognition domain. CNMT shows a similar overall fold (RMSD of 1.1 Å) to the recently determined structure of the pavine N ‐Methyltransferase (PavNMT) 9. CNMT forms a dimeric interface made up of 13 hydrogen bonds and 3 salt bridges, similar to that seen in the PavNMT structure.…”

supporting

confidence: 62%

“…The H208A mutant showed the most significant effect, resulting in very low enzyme activity of 1 and 4 % relative to wild‐type CNMT with substrates 8 and 3 respectively (Figure S6), suggesting H208 plays a key role in catalysis, most likely functioning as a general base to deprotonate the substrate ammonium ion. Sequence alignments with related NMTs including the PavNMT,9, 11 indicate that the H208 and E207 residues are highly conserved. Furthermore, mutation of these residues to alanine also results in a large decrease in the activity of PavNMT.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Structure and Biocatalytic Scope of Coclaurine N‐Methyltransferase

et al. 2018

View full text Add to dashboard Cite

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)‐reticulene at which point the pathway diverges. Coclaurine N‐methyltransferase (CNMT) is a key enzyme in the pathway to (S)‐reticulene, installing the N‐methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co‐factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives.

show abstract

supporting

confidence: 62%

mentioning

confidence: 99%

Structure and Biocatalytic Scope of Coclaurine N‐Methyltransferase

et al. 2018

View full text Add to dashboard Cite

show abstract

“…[ 31,32] The crystal structures of two MTase enzymes involved in benzylisoquinoline alkaloid biosynthesis have also recently been elucidated, the pavine-N-methyltransferase (PNMT) and norcoclaurine-6-O-methyltransferase (6OMT) ( Figure 2B). [33,34] The characterization of MTases from these pathways could be useful in developing biocatalytic approaches for production of alkaloid derivatives, which have been widely used as pharmaceuticals. Robin et al solved the structure of 6-OMT which is known to methylate at the 6-OH position of norcoclaurine producing coclaurine (14).…”

Section: )mentioning

confidence: 99%

Recent advances in methyltransferase biocatalysis

Bennett

Shepherd

Cronin

et al. 2017

Current Opinion in Chemical Biology

102

111

View full text Add to dashboard Cite

S-adenosyl-L-methionine-dependent methyltransferses are ubiquitous in nature, methylating a vast range of small molecule metabolites, as well as biopolymers. This review covers the recent advances in the development of methyltransferase enzymes for synthetic applications, focusing on the methyltransferase catalyzed transformations with S-adenosyl methionine analogs, as well as non-native substrates. We discuss how metabolic engineering approaches have been used to enhance S-adenosyl methionine production in vivo. Enzymatic approaches that enable the more efficient generation of S-adenosyl methionine analogs, including more stable analogs, will also be described; this has expanded the biocatalytic repertoire of methyltransferases from methylation to a broader range of alkylation reactions. The review also examines how the selectivity of the methyltransferase enzymes can be improved through structure guided mutagenesis approaches. Finally, we will discuss how methyltransferases can be deployed in multi-enzyme cascade reactions and suggest future challenges and avenues for further investigation.

show abstract

“…S4). In addition to the SAMbinding residues, which are entirely conserved, PaNMT also showed substantial conservation of residues predicted to form part of the homodimer interface and the alkaloid-binding domain in the TfPavNMT X-ray crystallographic structure (21). No canonical subcellular targeting signals were detected in the polypeptide sequence using TargetP (22).…”

Section: Characterization Of Panmtmentioning

confidence: 99%

An N-methyltransferase from Ephedra sinica catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production

Morris

Groves

Hagel

et al. 2018

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus involve-methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an -methyltransferase (NMT) from able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including -methylcathinone and-methyl(pseudo)ephedrine. Phenylalkylamine -methyltransferase (PaNMT) shares substantial amino acid sequence identity with enzymes of the NMT family involved in benzylisoquinoline alkaloid (BIA) metabolism in members of the higher plant order Ranunculales, which includes opium poppy (). PaNMT accepted a broad range of substrates with phenylalkylamine, tryptamine, β-carboline, tetrahydroisoquinoline, and BIA structural scaffolds, which is in contrast to the specificity for BIA substrates of NMT enzymes within the Ranunculales. PaNMT transcript levels were highest in young shoots of , which corresponded to the location of NMT activity yielding (pseudo)ephedrine,-methylcathinone, and -methyl(pseudo)ephedrine, and with accumulation of phenylalkylamines. Co-expression of recombinant genes encoding PaNMT and an ω-transaminase (PP2799) from in enabled the conversion of exogenous ()-phenylacetylcarbinol (PAC) and ()-PAC to ephedrine and pseudoephedrine, respectively. Our work further demonstrates the utility of plant biochemical genomics for the isolation of key enzymes that facilitate microbial engineering for the production of medicinally important metabolites.

show abstract

Structural and Functional Studies of Pavine N-Methyltransferase from Thalictrum flavum Reveal Novel Insights into Substrate Recognition and Catalytic Mechanism

Cited by 29 publications

References 34 publications

Structure and Biocatalytic Scope of Coclaurine N‐Methyltransferase

Structure and Biocatalytic Scope of Coclaurine N‐Methyltransferase

Recent advances in methyltransferase biocatalysis

An N-methyltransferase from Ephedra sinica catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production

Contact Info

Product

Resources

About