‘Dopamine‐first’ mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

Lichman, Benjamin R.; Gershater, Markus; Lamming, Eleanor D.; Pesnot, Thomas; Sula, Altin; Keep, Nicholas H.; Hailes, Helen C.; Ward, John M.

doi:10.1111/febs.13208

Cited by 66 publications

(147 citation statements)

References 34 publications

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“…A racemic mixture of norcoclaurine enantiomers produced non-enzymatically30 does not display a CD signal; thus, any spontaneous background condensation of dopamine and 4-HPAA will not affect kinetic values determined by CD spectroscopy. The measured K m values for dopamine and HPAA are consistent with our previous results710, but are substantially divergent from those in a recent report highlighting the inefficiency of NCS and potential solutions for metabolic engineering applications26. Comparison of the 4-HPAA and dopamine K cat values for CCHNCS2, containing two fused NCS domains, with those of NDONCS3 and TFLNCSΔ25, each possessing only single domains, is also in agreement with the proposed proportional increase in the catalytic efficiency of NCS fusions.…”

Section: Discussionsupporting

confidence: 68%

“…Despite playing a pivotal role in BIA biosynthesis, NCS is a relatively inefficient catalyst, which has been suggested as a salient feature considering its role as a ‘gatekeeper’ enzyme responsible for regulating entry into alkaloid metabolism26. However, NCS must still generate enough ( S )-norcoclaurine to support the production of abundant alkaloids, such as morphine, noscapine, and papaverine in opium poppy.…”

Section: Discussionmentioning

confidence: 99%

“…However, measured kinetic parameters for NCS suggest an enzyme that is catalytically inefficient67142526. NCS has been reported to display an apparent catalytic efficiency ( k cat / K m ) of 1.0 mM −1 s −1 , which is 100-fold lower than the median of 125 mM −1 s −1 calculated across all enzymes27, and a high apparent K m for dopamine interpreted as an indication that high cellular substrate concentrations are required for significant turnover26. The purported catalytic inefficiency of NCS could explain the relatively low turnover capacity of the enzyme in engineered microorganisms.…”

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confidence: 99%

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Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

Lee

Chang

et al. 2016

Sci Rep

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Norcoclaurine synthase (NCS) catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid (BIA) biosynthesis. NCS orthologs in available transcriptome databases were screened for variants that might improve the low yield of BIAs in engineered microorganisms. Databases for 21 BIA-producing species from four plant families yielded 33 assembled contigs with homology to characterized NCS genes. Predicted translation products generated from nine contigs consisted of two to five sequential repeats, each containing most of the sequence found in single-domain enzymes. Assembled contigs containing tandem domain repeats were detected only in members of the Papaveraceae family, including opium poppy (Papaver somniferum). Fourteen cDNAs were generated from 10 species, five of which encoded NCS orthologs with repeated domains. Functional analysis of corresponding recombinant proteins yielded six active NCS enzymes, including four containing either two, three or four repeated catalytic domains. Truncation of the first 25 N-terminal amino acids from the remaining polypeptides revealed two additional enzymes. Multiple catalytic domains correlated with a proportional increase in catalytic efficiency. Expression of NCS genes in Saccharomyces cereviseae also produced active enzymes. The metabolic conversion capacity of engineered yeast positively correlated with the number of repeated domains.

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

Lee

Chang

et al. 2016

Sci Rep

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“…[13,14,34] In this transition state,the aldehyde-derived substituent is pseudo-equatorial and oriented towards the active-site entrance while the methyl and hydroxy substituents are in pseudo-axial positions.I nt his scenario,t he constraints of the biocatalyst active-site architecture overwhelm any conformational preferences of the intermediate.…”

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confidence: 99%

“…Conversely,m ore sterically demanding 2-bromobenzaldehyde (6)isanexcellent cosubstrate in the stereoselective phosphate-catalyzed reaction;h owever, it is not accepted by NCS wild-type enzymes or variants thereof.F uture catalyst engineering of either phosphate or NCS would help increase the overlap of suitable aldehyde cosubstrates.S ynthesizing bulkier phosphate catalysts to improve the stereoselectivity for other aldehydes [35] and enzyme engineering of NCS to improve substrate tolerance towards bulkier aldehydes [14] could expand the range of this system further.…”

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confidence: 99%

Enzymatic and Chemoenzymatic Three‐Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines

et al. 2017

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Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in as tep-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves ac arboligation step,asubsequent transamination, and finally aP ictet-Spengler reaction with ac arbonyl cosubstrate.A ppropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol. Subsequent cyclization catalyzed either enzymatically by an orcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo-and biocatalysts for optimal results.The tetrahydroisoquinoline (THIQ) moiety is a" privileged scaffold", [1] and as such, it is found in numerous bioactive natural products (e.g., noscapine, [2] dioncophyllines [3] )a nd synthetic pharmaceutical drugs (e.g., solifenacin [4] ). THIQcontaining compounds demonstrate aw ide range of bioactivities,i ncluding antitumor, [2] antiparasitic, [3] and anticholinergic [4] properties. Naturally derived THIQs may be obtained by extraction or fermentation, [5] but in many cases,only small quantities are available,typically as one component of amixture.Chemical syntheses are af requent source of these compounds,b ut complex multistep asymmetric routes that give high stereoselectivities can be challenging,i ncluding at scale,a nd are often dependent on the use of toxic or environmentally harmful chemicals.[6] Therefore,n ovel synthetic routes towards THIQs are of significant interest.In vitro biocatalysis provides av iable method of producing complex THIQs in high stereoselectivities and under mild conditions. [7][8][9] Previous studies have shown norcoclaurine synthase (NCS), the Pictet-Spenglerase from plant benzylisoquinoline alkaloid biosynthesis, [10,11] to be av ersatile biocatalyst. NCS can convert 3-hydroxyphenethylamines (e.g.,d opamine) and ac arbonyl cosubstrate into a( 1 S)-THIQ with high stereoselectivities. [12][13][14][15] Thee nzyme has demonstrated ar emarkably wide carbonyl cosubstrate tolerance,a ccepting av ariety of aldehydes and ketones. [13][14][15][16][17][18][19][20] NCS has also been successfully employed in chemoenzymatic cascades. [12,15,16,21] As an alternative to NCS,i norganic phosphate can be used to catalyze the Pictet-Spengler reaction (PSR), typically forming racemic THIQs under aqueous conditions. [22] This can be successfully combined with enzyme steps. [21] Modular biocatalytic cascades [23] for the stereoselective production of pharmaceutical compounds are exemplified by the work conducted on phenylpropanolamines. [24][25][26] All four possible isomers of nor(pseudo)ephedrine are available from simple starting materials by one-pot two-enzym...

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Biochemistry, Genetics, and Genomics of Opium Poppy ( Papaver somniferum ) for Crop Improvement

Tamiru‐Oli

Premaratna

Gendall

et al. 2019

Annual Plant Reviews Online

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Opium poppy is the only commercially viable source of narcotic raw materials used by the alkaloid pharmaceutical industry. Considerable advances in our knowledge of basic poppy biology and the alkaloid biosynthetic pathway have been driven by recent progress in transcriptomics, genomics, and functional genetics. However, much work remains for this knowledge to be translated into improvements in crop performance. The genetic diversity of poppy is poorly characterised and the available information is highly fragmented. The recent release of a poppy genome sequence adds a new dimension to poppy genomic research, enabling characterisation of diversity and identification of genes and molecular markers associated with valuable traits. This will create opportunities for functional genomics studies and the incorporation of more diverse germplasm into poppy improvement programmes. This review discusses the current state of poppy genetic and genomic resources, highlights the advances made in elucidating the alkaloid biosynthetic pathway that led to the emergence of poppy as a model system to study secondary metabolism in plants, and presents perspectives for future research.

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‘Dopamine‐first’ mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile

Cited by 66 publications

References 34 publications

Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

Enzymatic and Chemoenzymatic Three‐Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines

Biochemistry, Genetics, and Genomics of Opium Poppy ( Papaver somniferum ) for Crop Improvement

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