Understanding the Enzyme (S)-Norcoclaurine Synthase Promiscuity to Aldehydes and Ketones.

The (S)-norcoclaurine synthase from Thalictrum flavum (TfNCS) stereoselectively catalyzes the Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. TfNCS can catalyze the Pictet-Spengler reaction with various aldehydes and ketones, leading to diverse tetrahydroisoquinolines. This substrate promiscuity positions TfNCS as a highly promising enzyme for synthesizing fine chemicals. Understanding carbonyl-containing substrates' structural and electronic signatures that influence TfNCS activity can help expand its applications in the synthesis of different compounds and aid in protein optimization strategies. In this study, we investigated the influence of the molecular properties of aldehydes and ketones on their reactivity in the TfNCS-catalyzed Pictet-Spengler reaction. Initially, we compiled a library of reactive and unreactive compounds from previous publications. We also performed enzymatic assays using nuclear magnetic resonance to identify some reactive and unreactive carbonyl compounds, which were then included in the library. Subsequently, we employed QSAR and DFT calculations to establish correlations between substrate-candidate structures and reactivity. Our findings highlight correlations of structural and stereoelectronic features, including the electrophilicity of the carbonyl group, to the reactivity of aldehydes and ketones toward the TfNCS-catalyzed Pictet-Spengler reaction. Interestingly, experimental data of seven compounds out of fifty-three did not correlate with the electrophilicity of the carbonyl group. For these seven compounds, we identified unfavorable interactions between them and the TfNCS. Our results demonstrate the applications of in silico techniques in understanding enzyme promiscuity and specificity, with a particular emphasis on machine learning methodologies, DFT electronic structure calculations, and molecular dynamic (MD) simulations.

Structural Evidence for the Dopamine-First Mechanism of Norcoclaurine Synthase.

Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS "dopamine-first" mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst.

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

Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet-Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine 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.

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

Benzylisoquinoline alkaloids (BIAs) are produced in a wide variety of plants and include many common analgesic, antitussive, and anticancer compounds. Several members of a distinct family of S-adenosylmethionine (SAM)-dependent N-methyltransferases (NMTs) play critical roles in BIA biosynthesis, but the molecular basis of substrate recognition and catalysis is not known for NMTs involved in BIA metabolism. To address this issue, the crystal structure of pavine NMT from Thalictrum flavum was solved using selenomethionine-substituted protein (dmin = 2.8 Å). Additional structures were determined for the native protein (dmin = 2.0 Å) as well as binary complexes with SAM (dmin = 2.3 Å) or the reaction product S-adenosylhomocysteine (dmin = 1.6 Å). The structure of a complex with S-adenosylhomocysteine and two molecules of tetrahydropapaverine (THP; one as the S conformer and a second in the R configuration) (dmin = 1.8 Å) revealed key features of substrate recognition. Pavine NMT converted racemic THP to laudanosine, but the enzyme showed a preference for (±)-pavine and (S)-reticuline as substrates. These structures suggest the involvement of highly conserved residues at the active site. Mutagenesis of three residues near the methyl group of SAM and the nitrogen atom of the alkaloid acceptor decreased enzyme activity without disrupting the structure of the protein. The binding site for THP provides a framework for understanding substrate specificity among numerous NMTs involved in the biosynthesis of BIAs and other specialized metabolites. This information will facilitate metabolic engineering efforts aimed at producing medicinally important compounds in heterologous systems, such as yeast.

Crystal structure of norcoclaurine-6-O-methyltransferase, a key rate-limiting step in the synthesis of benzylisoquinoline alkaloids.

Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate-limiting enzymes and negative feedback inhibition by end-products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)-norcoclaurine-6-O-methyltransferase (6OMT), a key rate-limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end-products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S-adenosyl-l-homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time-dependent sensitivity toward sanguinarine.

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine.

Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family.

Norcoclaurine synthase (NCS) catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids (BIAs). NCS from Thalictrum flavum (Tf NCS), Papaver somniferum (Ps NCS1 and Ps NCS2), and Coptis japonica (Cj PR10A) share substantial identity with pathogen-related 10 (PR10) and Bet v1 proteins, whose functions are not well understood. A distinct enzyme (Cj NCS1) with similarity to 2-oxoglutarate-dependent dioxygenases was suggested as the bona fide NCS in C. japonica. Here, we validate the exclusive role of PR10/Bet v1-type NCS enzymes in BIA metabolism. Immunolocalization of Ps NCS2 revealed its cell type-specific occurrence in phloem sieve elements, which contain all other known BIA biosynthetic enzymes. In opium poppy, NCS transcripts and proteins were abundant in root and stem, but at low levels in leaf and carpel. Silencing of NCS in opium poppy profoundly reduced alkaloid levels compared with controls. Immunoprecipitation of NCS from total protein extracts of T. flavum cells resulted in a nearly complete attenuation of NCS activity. A Ps NCS2-green fluorescent protein fusion introduced by microprojectile bombardment into opium poppy cells initially localized to the endoplasmic reticulum but subsequently sorted to the vacuole. In our hands, Cj NCS1 did not catalyze the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde.

Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species.

An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant-specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI-FTICR-MS and ESI-MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N-methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N-methyltransferase (TfPavNMT), which showed a unique preference for (+/-)-pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species-dependent specialized metabolite profiles.

Structural basis of enzymatic (S)-norcoclaurine biosynthesis.

The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1A resolution. NCS shares no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-A-long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.

Purification, crystallization and X-ray diffraction analysis of pavine N-methyltransferase from Thalictrum flavum.

A cDNA from the plant Thalictrum flavum encoding pavine N-methyltransferase, an enzyme belonging to a novel class of S-adenosylmethionine-dependent N-methyltransferases specific for benzylisoquinoline alkaloids, has been heterologously expressed in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatography and was crystallized in space group P2(1). The structure was solved at 2.0 A resolution using a xenon derivative and the single isomorphous replacement with anomalous scattering method.

Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum.

Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 A resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 86.31, c = 118.36 A. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 A resolution. The model is under construction.

High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies.

The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure.

Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: an enzymatic Pictet-Spengler reaction.

Norcoclaurine synthase catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a kinetic isotope effect of 1.7 +/- 0.1 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing rearomatization is partially rate determining in the overall reaction.

Cell type-specific localization of transcripts encoding nine consecutive enzymes involved in protoberberine alkaloid biosynthesis.

Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of l-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type-specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type-specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types.

Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis.

(S)-Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids such as morphine, sanguinarine, and berberine, in plants. A molecular clone encoding NCS was isolated from a meadow rue (Thalictrum flavum ssp. glaucum) cell suspension culture cDNA library. Heterologous expression of the NCS cDNA, truncated to remove a putative signal peptide, produced a recombinant protein with NCS activity. Recombinant NCS showed sigmoidal saturation kinetics for dopamine (Hill coefficient=1.98), hyperbolic saturation kinetics for 4-HPAA (Km of 700 microm), and pH and temperature optima of 7.0 and 40 degrees C, respectively, all similar to the purified, plant-derived enzyme. NCS exhibits 28-38% identity, and putative structural homology, with the Bet v 1 allergen and pathogenesis-related (PR)10 protein families. NCS also displays 35% identity with the enzyme (HYP1) responsible for hypericin biosynthesis in St John's wort (Hypericum perforatum). The novel catalytic functions of NCS and HYP1 define a new class of plant secondary metabolic enzymes within the Bet v 1 and PR10 protein families. Weaker homology was also detected between NCS and proteins identified in the latex of Papaver somniferum (opium poppy), and in Arabidopsis thaliana. A family of three to five NCS genes is abundantly expressed in the rhizome, followed by petioles and roots of T. flavum. NCS transcripts were localized to the immature endodermis and pericycle in roots, and the protoderm of leaf primordia in rhizomes; thus, the sites of NCS gene expression and berberine accumulation are temporally and spatially separated in roots and rhizomes respectively.

Purification and characterization of norcoclaurine synthase. The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants.

Norcoclaurine synthase (NCS; EC ) catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in benzylisoquinoline alkaloid biosynthesis in plants. NCS was purified 1590-fold to homogeneity from cell suspension cultures of meadow rue (Thalictrum flavum ssp. glaucum). The purification procedure, which resulted in a 4.2% yield, involved hydrophobic interaction, anion exchange, hydroxyapatite, and gel filtration chromatography. Purified NCS displayed native and denatured molecular masses of approximately 28 and 15 kDa, respectively, suggesting that the enzyme is composed of two subunits. Two-dimensional polyacrylamide gel electrophoresis revealed two major and two minor isoforms with pI values between 5.5 and 6.2. NCS activity was maximal at pH 6.5 to 7.0 and temperatures between 42 and 55 degrees C and was not affected by divalent cations. The enzyme showed hyperbolic saturation kinetics for 4-HPAA (K(m) = 335 microm) but sigmoidal saturation kinetics for dopamine (Hill coefficient = 1.8) suggesting cooperativity between the dopamine binding sites on each subunit; thus, NCS might play a regulatory, or rate-limiting, role in controlling the rate of pathway flux in benzylisoquinoline alkaloid biosynthesis. Product inhibition kinetics performed at saturating levels of one substrate and with norlaudanosoline as the inhibitor showed that NCS follows an iso-ordered bi-uni mechanism with 4-HPAA binding before dopamine. NCS activity was highest in soluble protein extracts from roots followed by stems, leaves, and flower buds.