Acceptance and Kinetic Resolution of α-Methyl-Substituted Aldehydes by Norcoclaurine Synthases

Roddan, Rebecca; Gygli, Gudrun; Sula, Altin; Méndez-Sánchez, Daniel; Pleiss, Jürgen; Ward, John M.; Keep, Nicholas H.; Hailes, Helen C.

doi:10.1021/acscatal.9b02699

Cited by 33 publications

(74 citation statements)

References 33 publications

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“…In previous screens, when dopamine and benzaldehyde were used with TfNCS, products were not detected or trace conversions were observed, possibly due to the lower enzyme or substrate loading used 25 . As demonstrated in previous reports, high substrate loading is well-tolerated by NCS and is indeed required to gain high conversions of the substrate (or high yields of product) 23,24 . The K d value of dopamine was determined by NMR titration experiments and found to be 5 mM, thus indicating very weak substrate binding 27 .…”

Section: Resultsmentioning

confidence: 72%

“…30 and Supplementary Methods), and a truncated construct of M97V-TfNCS generated, M97V-Δ33TfNCS, omitting residues (1-33 and 196-210; Supplementary Figs. 1-3 and Supplementary Methods) in the signal peptides at both termini, as with previous studies 23,38 . A co-crystallised structure was gained of M97V-Δ33TfNCS with 6 bound in the active site ( Fig.…”

Section: Resultsmentioning

confidence: 73%

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Single step syntheses of (1S)-aryl-tetrahydroisoquinolines by norcoclaurine synthases

et al. 2020

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The 1-aryl-tetrahydroisoquinoline (1-aryl-THIQ) moiety is found in many biologically active molecules. Single enantiomer chemical syntheses are challenging and although some biocatalytic routes have been reported, the substrate scope is limited to certain structural motifs. The enzyme norcoclaurine synthase (NCS), involved in plant alkaloid biosynthesis, has been shown to perform stereoselective Pictet–Spengler reactions between dopamine and several carbonyl substrates. Here, benzaldehydes are explored as substrates and found to be accepted by both wild-type and mutant constructs of NCS. In particular, the variant M97V gives a range of (1 S)-aryl-THIQs in high yields (48–99%) and e.e.s (79–95%). A co-crystallised structure of the M97V variant with an active site reaction intermediate analogue is also obtained with the ligand in a pre-cyclisation conformation, consistent with (1 S)-THIQs formation. Selected THIQs are then used with catechol O-methyltransferases with exceptional regioselectivity. This work demonstrates valuable biocatalytic approaches to a range of (1 S)-THIQs.

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Section: Resultsmentioning

confidence: 72%

Section: Resultsmentioning

confidence: 73%

Single step syntheses of (1S)-aryl-tetrahydroisoquinolines by norcoclaurine synthases

et al. 2020

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“…3b). Analogous substituted THIQs derived from catabolism of L-isoleucine and L-valine were not observed since α-substituted aldehydes are not well tolerated by NCS 38 . Targeted fragmentation of substituted THIQ products yielded spectra consistent with the presumed metabolite identities 39 ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids

et al. 2020

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The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate (S)-reticuline to 4.6 g L −1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

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“…The mechanism of NCS (Scheme 24C) has been elucidated through structural 182,183 and computational analyses. 147 Dopamine binds in the enzyme active site prior to 4-HPAA, with the catechol 3-OH H-bonding to the Lys-122 residue deep in the active site and the residues Glu-110 and Asp-141 interact with the dopamine amine group.…”

Section: Aromatic Amino-acid Derivedmentioning

confidence: 99%

“…148 Iminium formation probably involves acid/base catalysis from Glu-110 and Asp-141, and may also involve changes to the enzyme structure. 183 The key steps of C-C bond formation and subsequent loss of proton are catalysed by Lys-122 and Glu-110 respectively.…”

Section: Aromatic Amino-acid Derivedmentioning

confidence: 99%