Radioimmunoassay determination of six opium alkaloids and its application to plant screening

Radioimmunoassay procedures were developed for the independent and specific determination of sub-nmole quantities of (S)- and (R)-reticuline, salutaridine, thebaine, codeine and morphine. Assay parameters for all six poppy alkaloids are given and the synthesis of haptens and tracers in the Ci/mmol range is described. These assays were used to screen herbarium material of 100 Papaver species and to analyse P. somniferum plant populations for alkaloid breeding purposes. The time course of alkaloid appearance during the germination of poppy seeds was also studied.     

Biotransformation of (RS)-reticuline and morphinan alkaloids by cell cultures of Papaver somniferum

(RS)-Reticuline was stereospecifically converted to (—)-(S)-scoulerine and (—)-(S)-cheilanthifoline by cell cultures of Papaver somniferum and (—)-(R)-reticuline was recovered as an optical pure compound by racemic resolution. (—)-Codeinone was converted in high yield to (—)-codeine in both cell culture and enzyme preparation, but the other morphinans, thebaine, codeine and morphine, were not metabolized.     

CYP719B1 Is Salutaridine Synthase, the C-C Phenol-coupling Enzyme of Morphine Biosynthesis in Opium Poppy

Morphine is a powerful analgesic natural product produced by the opium poppy Papaver somniferum. Although formal syntheses of this alkaloid have been reported, the morphine molecule contains five stereocenters and a C-C phenol linkage that to date render a total synthesis of morphine commercially unfeasible. The C-C phenol-coupling reaction along the biosynthetic pathway to morphine in opium poppy is catalyzed by the cytochrome P450-dependent oxygenase salutaridine synthase. We report herein on the identification of salutaridine synthase as a member of the CYP719 family of cytochromes P450 during a screen of recombinant cytochromes P450 of opium poppy functionally expressed in Spodoptera frugiperda Sf9 cells. Recombinant CYP719B1 is a highly stereo- and regioselective enzyme; of forty-one compounds tested as potential substrates, only (R)-reticuline and (R)-norreticuline resulted in formation of a product (salutaridine and norsalutaridine, respectively). To date, CYP719s have been characterized catalyzing only the formation of a methylenedioxy bridge in berberine biosynthesis (canadine synthase, CYP719A1) and in benzo[c]phenanthridine biosynthesis (stylopine synthase, CYP719A14). Previously identified phenol-coupling enzymes of plant alkaloid biosynthesis belong only to the CYP80 family of cytochromes. CYP719B1 therefore is the prototype for a new family of plant cytochromes P450 that catalyze formation of a phenol-couple.The C-O or C-C phenol-couple is widely present in the plant kingdom in natural product biosynthetic processes such as alkaloid (1), lignan (2), lignin (3), and gallotannin (4) formation. Phenol-coupling reactions in nature were thought to be catalyzed by a variety of oxidative enzymes with broad substrate specificity such as peroxidases, polyphenol oxidases, and laccases. More recently, several enzymes discovered to be responsible for the formation of intermolecular C-O phenol and intramolecular C-C phenol-couples were found to be highly regio- and/or stereoselective catalysts. The first intermolecular C-O phenol-coupling enzyme identified was the cytochrome P450-dependent oxidase berbamunine synthase (CYP80A1) of bisbenzylisoquinoline alkaloid biosynthesis in Berberis cell cultures (5, 6) (Fig. 1). This enzyme is regiospecific, but will accept either (R)- and (S)-N-methylcoclaurine to form R-R and R-S phenol-coupled products. Absolute regio- and stereospecificity is demonstrated in the formation of the lignan (+)-pinoresinol from two molecules of coniferyl alcohol, a reaction guided by dirigent proteins that can be catalyzed by a range of oxidases or oxidants (7). The aporphine alkaloid intramolecular C-C phenol-couple is catalyzed in Coptis japonica cell cultures by the cytochrome P450-dependent oxidase CYP80G2; this enzyme accepts six tetrahydrobenzylisoquinoline alkaloids as substrate (8).Open in a separate windowFIGURE 1.Selected phenol-coupling reactions of alkaloid biosynthesis. Berbamunine synthase (CYP80A1) catalyzes the C-O intermolecular phenol-coupling reaction of bisbenzyisoquinoline alkaloid biosynthesis. (S)-Corytuberine synthase (CYP80G2) catalyzes formation of the intramolecular C-C phenol-couple in magnoflorine biosynthesis. Salutaridine synthase forms the C-C intramolecular phenol-couple of salutaridine in morphine biosynthesis.Morphine has often been described as the king of alkaloids. Although formal syntheses of this powerful analgesic have been reported, yields are low (Ref. 9 and references therein); attempts in organic chemistry to mimic the biosynthetic formation of the C-C phenol-couple of salutaridine (Fig. 1) have been either unsuccessful, yielding rather isoboldine or pallidine (10), or have resulted in very low yield of salutaridine (11) or in a mixture of isoboldine and salutaridine, with the reaction favoring formation of isoboldine by a factor of ∼5 (12). Along with the five stereocenters present in this molecule, the C-C phenol-couple renders a chemical synthesis of morphine commercially unfeasible. The enzyme catalyzing this reaction in planta was sought unsuccessfully for many years and was discovered finally in the opium poppy Papaver somniferum to be a cytochrome P450-dependent oxidase that stereo- and regiospecifically produces salutaridine by C-C phenol-coupling of (R)-reticuline (Fig. 1) (1, 13). The native enzyme salutaridine synthase was unstable, which precluded protein purification for further characterization.Herein, we describe the identification and functional expression of opium poppy salutaridine synthase, a member of the cytochrome P450 family, in Spodoptera frugiperda Sf9 cells. The recombinant enzyme was sufficiently stable in insect cell culture to be characterized with respect to substrate specificity and steady state kinetic values. Recombinant salutaridine synthase converted (R)-reticuline exclusively to salutaridine and (R)-norreticuline exclusively to norsalutaridine (N-demethylsalutaridine).     

Mammalian Cytochrome P450 Enzymes Catalyze the Phenol-coupling Step in Endogenous Morphine Biosynthesis

A cytochrome P450 (P450) enzyme in porcine liver that catalyzed the phenol-coupling reaction of the substrate (R)-reticuline to salutaridine was previously purified to homogeneity (Amann, T., Roos, P. H., Huh, H., and Zenk, M. H. (1995) Heterocycles 40, 425–440). This reaction was found to be catalyzed by human P450s 2D6 and 3A4 in the presence of (R)-reticuline and NADPH to yield not a single product, but rather (−)-isoboldine, (−)-corytuberine, (+)-pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppy plant and most likely also in mammals. (S)-Reticuline, a substrate of both P450 enzymes, yielded the phenol-coupled alkaloids (+)-isoboldine, (+)-corytuberine, (−)-pallidine, and sinoacutine; none of these serve as a morphine precursor. Catalytic efficiencies were similar for P450 2D6 and P450 3A4 in the presence of cytochrome b₅ with (R)-reticuline as substrate. The mechanism of phenol coupling is not yet established; however, we favor a single cycle of iron oxidation to yield salutaridine and the three other alkaloids from (R)-reticuline. The total yield of salutaridine formed can supply the 10 nm concentration of morphine found in human neuroblastoma cell cultures and in brain tissues of mice.Cytochrome P450 (P450)² enzymes catalyze the most versatile chemical reactions in nature (1). There is, however, a discrepancy between the plant and the animal kingdoms with regard to the sheer number of these biocatalysts. Whereas in a single model plant, Arabidopsis thaliana, there are to date 273 P450 proteins, in the human genome, only 57 of these proteins are present. Whereas plants and animals share a multitude of highly regio- and stereospecific O-demethylation reactions, more complex reactions such as phenol coupling are much more abundant in plants than in animals, especially in the alkaloid field (2–11). The proposal of Barton and Cohen (12) correlated the structure of specific plant alkaloids in terms of this reaction mechanism and gave mechanistic proposals of how these phenol-coupled products may possibly be biosynthesized in nature. The oxidation of phenols by one-electron transfer affords radicals, which, by radical pairing, form new C-C or C-O bonds either by intra- or intermolecular coupling. The first two examples that unequivocally demonstrated the formation of C-C and C-O bonds in a stereo- and regioselective manner in plant metabolism are catalyzed by specific P450-linked microsomal-bound plant enzymes (13). One of these enzymes was salutaridine synthase from Papaver somniferum (opium poppy) (14). This synthase catalyzes the intramolecular formation of the critical C12-C13 carbon bridge and is a key enzyme in morphine biosynthesis.The groups of Goldstein (15) and Spector (16) have published a number of reports over the past 25 years claiming that mammals are capable of synthesizing de novo traces of endogenous morphine. However, no convincing experimental data have been presented regarding the enzymes. Phenol-coupling reactions in mammals are extremely rare, and the only example described thus far is the formation of thyroxine, by radical pairing, in humans. If the key step of morphine synthesis, the formation of phenol-coupled salutaridine from (R)-reticuline, occurs in mammals then in analogy to plants, a P450 enzyme must be present (in mammals) to catalyze this reaction. In 1987, the first experiments were conducted in an attempt to discover the reaction by supplying uniformly labeled racemic [³H]reticuline in the presence of rat microsomes and NADPH to examine whether [³H]salutaridine can be formed under these conditions (15). A radioactive compound was formed in 1% yield and assumed to be the phenol-coupled product salutaridine. We later repeated this experiment using (R)-[N-¹⁴CH₃]reticuline and NADPH-fortified microsomes from pig, rat, cow, and sheep. We observed the formation of [N-¹⁴CH₃]salutaridine with the correct stereochemistry at carbon 9 (17). The enzyme from porcine liver was subsequently purified to homogeneity and the reaction product was characterized by mass spectrometry and physical parameters to be a product of a P450 enzyme, which we provisionally named “salutaridine synthase” (18). The aim of this report is the identification of the homogenous porcine P450 enzyme, its equivalent in humans, and the mechanism of the phenol-coupling reaction in the formation of precursors of morphine.     

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel α-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.The benzylisoquinoline alkaloids (BIAs)³ comprise a large and diverse group of nitrogen-containing secondary metabolites with about 2500 compounds identified in plants (1). Among them are several important pharmaceuticals, such as the antimicrobials berberine and sanguinarine, and the vasodilator papaverine. The most prominent compounds of this class are the antitussive codeine, the analgesic morphine, and their biosynthetic precursor thebaine. The latter is used as the starting molecule for the production of a variety of semi-synthetic analgesics including oxycontin and buprenorphine. Pentacyclic morphinan alkaloids possess several chiral centers, which preclude chemical synthesis as an option for the efficient production of these widely used pharmaceuticals. Therefore, the worldwide supply of these narcotic compounds is still achieved by their isolation mainly from the opium poppy, Papaver somniferum L. With the availability of an increasing number of isolated genes encoding several pathway enzymes, recent interest has focused on the qualitative and quantitative modulation of the alkaloid profile in transgenic opium poppy plants (2–6), the production of BIAs in microbes (7, 8), and de novo synthesis by a combination of chemical and biochemical conversions. BIA biosynthesis begins with the condensation of the tyrosine-derived precursors dopamine and p-hydroxyphenylacetaldehyde to (S)-norcoclaurine (see Fig. 1) (1). Subsequent regiospecific O- and N-methylations and aromatic ring hydroxylation lead to (S)-reticuline, which is the central intermediate for almost all BIAs. For morphinan alkaloid biosynthesis, (S)-reticuline undergoes an inversion of stereochemistry to (R)-reticuline, followed by C-C phenol coupling catalyzed by a unique cytochrome P450-dependent monooxygenase to yield salutaridine. Subsequent stereospecific reduction to 7(S)-salutaridinol is required for the attachment of an acetyl moiety to produce salutaridinol-7-O-acetate, which spontaneously rearranges to thebaine (9). The O-demethylation of thebaine and the reduction of codeinone to codeine represent the penultimate steps in morphine biosynthesis. Cognate cDNAs have been isolated for all of the enzymes leading to (S)-reticuline, as well as those involved in the conversion of (R)-reticuline to salutaridine-7-O-acetate (1). Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific, NADPH-dependent reduction of salutaridine to 7(S)-salutaridinol and is a member of the classical subgroup of the short chain dehydrogenase/reductase (SDR) protein family (10, 11). The main characteristics of this category of SDRs are the largely conserved TGXXXGhG motif for cofactor binding and the YXXXK motif, which together with an upstream Ser residue represent the catalytic center (12). In this catalytic triad, Lys forms hydrogen bonds with the ribose moiety of the cofactor, which itself is hydrogen bonded to Tyr. This hydrogen bond network is presumed to lower the pK_a of the Tyr hydroxyl group, which functions as the catalytic base. Ser has been suggested to either stabilize the substrate (13, 14) or to interact with Tyr (15). Additionally, an Asn residue has been proposed to stabilize the position of the Lys residue, thereby forming a proton relay system involving water (16). Most other members of the SDR protein family are categorized into three additional subgroups (i.e. divergent, intermediate, or complex) exhibiting different overall sizes and slight amino acid sequence variations in conserved regions (17). Non-classical SDRs predominantly consist of isomerases (EC 5.-.-.-), such as galactose epimerase, and lyases (EC 4.-.-.-), such as glucose dehydratase, whereas classical SDRs encompass oxidoreductases (EC 1.-.-.-), such as SalR. Although classical SDRs are typically multimeric, SalR is a monomer because of an additional stretch of 40 amino acids preceding the YXXXK catalytic motif. In porcine testicular carbonyl reductase, these amino acids form a helix blocking the dimer interface (18) and homology modeling revealed a similar feature in SalR (19). In contrast with porcine testicular carbonyl reductase, SalR exhibits an additional stretch of 40 amino acids that have only been detected in some SDRs from plants (11, 20–22). Although attempts to obtain a crystal structure for SalR have so far been unsuccessful, homology modeling using porcine testicular carbonyl reductase as a template produced a tertiary structure in which the additional amino acids form an additional helix (19). The subsequent docking of salutaridine into the active site of this model suggested the involvement of this structural element in substrate binding, which was supported by preliminary site-directed mutagenesis.Open in a separate windowFIGURE 1.Selected steps in morphine biosynthesis. Double arrows indicate the involvement of more than one enzyme. The SalR reaction is highlighted. HPAA, p-hydroxyphenylacetaldehyde; SalR, salutaridine reductase.SalR from the Persian poppy Papaver bracteatum L. shows strong substrate inhibition with a K_i around 150 μm (19). Substrate inhibition can substantially and negatively impact chemical engineering strategies by limiting the quantity of substrate that can be fed into a system, which reduces overall efficiency. The strong substrate inhibition exhibited by SalR could limit its biotechnological application in plant, microbial, or enzyme-based systems. To investigate the structural basis of substrate inhibition, we substituted all amino acids putatively involved in salutaridine binding and analyzed various kinetic parameters. Over the course of these experiments, substrate docking required modification because some mutations had unexpected consequences that did not fully agree with the original docking. The discrepancy was mainly due to the side chain arrangements of amino acids residing in the new helix. Precise prediction of this domain is difficult because of the lack of an equivalent crystal structure. In this report, we present a revised substrate docking for salutaridine into SalR, supported by comprehensive site-directed mutagenesis, which facilitated the creation of an enzyme variant devoid of substrate inhibition.     

Morphine Biosynthesis in Opium Poppy Involves Two Cell Types: Sieve Elements and Laticifers

Immunofluorescence labeling and shotgun proteomics were used to establish the cell type–specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.     

Complementary microbial approaches for the preparation of optically pure aromatic molecules

Different strategies for stereoselective microbial preparation of various chiral aromatic compounds are described. Optically pure 2-methyl-3-phenyl-1-propanol, ethyl 2-methyl-3-phenylpropanoate, 2-methyl-3-phenylpropanal, 2-methyl-3-phenylpropionic acid and 2-methyl-3-phenylpropyl acetate have been prepared using different microbial biotransformations starting from different prochiral and/or racemic substrates. (S)-2-Methyl-3-phenyl-1-propanol and (S)-2-methyl-3-phenylpropanal were prepared by biotransformation of 2-methyl cinnamaldehyde using the recombinant strain Saccharomyces cerevisiae BY4741ΔOye2Ks carrying a heterologous OYE gene from Kazachstania spencerorum. (R)-2-Methyl-3-phenylpropionic acid was obtained by oxidation of racemic 2-methyl-3-phenyl-1-propanol with acetic acid bacteria. Kinetic resolution of racemic 2-methyl-3-phenylpropionic acid was carried out by direct esterification with ethanol using dry mycelia of Rhizopus oryzae CBS 112.07 in organic solvent, giving (R)-ethyl 2-methyl-3-phenylpropanoate as major enantiomer. Finally, (R,S)-2-methyl-3-phenylpropyl acetate was enantioselectively hydrolysed employing different bacteria and yeasts having cell-bound carboxylesterases with prevalent formation of (R)- or (S)-2-methyl-3-phenyl-1-propanol, depending on the strain employed.     

Deletion of the GRE3 Aldose Reductase Gene and Its Influence on Xylose Metabolism in Recombinant Strains of Saccharomyces cerevisiae Expressing the xylA and XKS1 Genes

Saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. When the bacterial enzyme xylose isomerase (XI) from Thermus thermophilus was produced in S. cerevisiae, xylose utilization and ethanol formation were demonstrated. In addition, xylitol and acetate were formed. An unspecific aldose reductase (AR) capable of reducing xylose to xylitol has been identified in S. cerevisiae. The GRE3 gene, encoding the AR enzyme, was deleted in S. cerevisiae CEN.PK2-1C, yielding YUSM1009a. XI from T. thermophilus was produced, and endogenous xylulokinase from S. cerevisiae was overproduced in S. cerevisiae CEN.PK2-1C and YUSM1009a. In recombinant strains from which the GRE3 gene was deleted, xylitol formation decreased twofold. Deletion of the GRE3 gene combined with expression of the xylA gene from T. thermophilus on a replicative plasmid generated recombinant xylose utilizing S. cerevisiae strain TMB3102, which produced ethanol from xylose with a yield of 0.28 mmol of C from ethanol/mmol of C from xylose. None of the recombinant strains grew on xylose.     

Conversion of codeine to morphine in isolated capsules of Papaver somniferum

The biotransformation of codeine to morphine was studied in isolated capsules of Papaver somniferum. Cofactors such as nicotinamide adenine dinucleotide, adenosine 5′-triphosphate, S-acetyl coenzyme A and pyridoxal phosphate were not required in the conversion of codeine to morphine. Reducing agents such as dithiothreitol, glutathione and β-mercaptoethanol strongly promoted codeine and morphine degradation, while morphine formation remained at a constant level. Hydrogen peroxide (concentration > 0.25 mM) caused the conversion of codeine and morphine to N-oxides by non-enzymatic oxidation. Isolated capsules of P. somniferum provide a method of studying the biotransformation of codeine to morphine.     

Laboratory-scale production of (S)-reticuline,an important intermediate of benzylisoquinoline alkaloids,using a bacterial-based method

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery.     

Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–¹³C₆ glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron–sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron–sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron–sulfur cluster proteins in its cytosol.     

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Atomic structure of salutaridine reductase from the opium poppy (Papaver somniferum)

The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of ∼1.9 Å in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265–279), on top of which lies a large “flap”-like domain (residues 105–140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.     

Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L⁻¹ of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L⁻¹ h⁻¹ g⁻¹ wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase.     

An enzymatic approach to the synthesis of optically pure (3R)- and (3S)-enantiomers of green tea flavor compound 3-hydroxy-3-methylnonane-2,4-dione

Both (3R)- and (3S)-enantiomers of the chiral green tea flavor compound 3-hydroxy-3-methylnonane-2,4-dione were synthesized by the combined use of acetylacetoin synthase and acetylacetoin reductase from Bacillus licheniformis. The first enzyme was utilized to catalyze the homo-coupling of 2,3-octanedione and obtain the enantioenriched (3R)-3-hydroxy-3-methylnonane-2,4-dione (ee 44%). The NADH-dependent acetylacetoin reductase was then employed for the diastereoselective (de > 95%) C2 carbonyl reduction of the sole (3R)-enantiomer of the above 2,4-dione, thus affording the syn diol (2S,3R)-2,3-dihydroxy-3-methylnonan-4-one in enantiomerically pure form. While this step allowed for the recovery of unreacted, optically pure (3S)-3-hydroxy-3-methylnonae-2,4-dione, the corresponding (3R)-enantiomer was obtained by subsequent TEMPO-mediated oxidation of the syn diol intermediate. Moreover, using the title compounds as analytical standards, predominance of the (3R) enantiomer in the natural flavor compound was finally demonstrated by chiral GC–MS analysis.     

High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate

Acetoin is widely used in food and cosmetics industries as a taste and fragrance enhancer. To produce (R)-acetoin in Saccharomyces cerevisiae, acetoin biosynthetic genes encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) from Bacillus subtilis and water-forming NADH oxidase (NoxE) from Lactococcus lactis were integrated into delta-sequences in JHY605 strain, where the production of ethanol, glycerol, and (R,R)-2,3-butanediol (BDO) was largely eliminated. We further improved acetoin production by increasing acetoin tolerance by adaptive laboratory evolution, and eliminating other byproducts including meso-2,3-BDO and 2,3-dimethylglycerate, a newly identified byproduct. Ara1, Ypr1, and Ymr226c (named Ora1) were identified as (S)-alcohol-forming reductases, which can reduce (R)-acetoin to meso-2,3-BDO in vitro. However, only Ara1 and Ypr1 contributed to meso-2,3-BDO production in vivo. We elucidate that Ora1, having a substrate preference for (S)-acetoin, reduces (S)-α-acetolactate to 2,3-dimethylglycerate, thus competing with AlsD-mediated (R)-acetoin production. By deleting ARA1, YPR1, and ORA1, 101.3 g/L of (R)-acetoin was produced with a high yield (96% of the maximum theoretical yield) and high stereospecificity (98.2%).