Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.

Identification of a drimenol synthase and drimenol oxidase from Persicaria hydropiper, involved in the biosynthesis of insect deterrent drimanes.

The sesquiterpenoid polygodial, which belongs to the drimane family, has been shown to be an antifeedant for a number of herbivorous insects. It is presumed to be synthesized from farnesyl diphosphate via drimenol, subsequent C-12 hydroxylation and further oxidations at both C-11 and C-12 to form a dialdehyde. Here, we have identified a drimenol synthase (PhDS) and a cytochrome P450 drimenol oxidase (PhDOX1) from Persicaria hydropiper. Expression of PhDS in yeast and plants resulted in production of drimenol alone. Co-expression of PhDS with PhDOX1 in yeast yielded drimendiol, the 12-hydroxylation product of drimenol, as a major product, and cinnamolide. When PhDS and PhDOX1 were transiently expressed by agro-infiltration in Nicotiana benthamiana leaves, drimenol was almost completely converted into cinnamolide and several additional drimenol derivatives were observed. In vitro assays showed that PhDOX1 only catalyses the conversion from drimenol to drimendiol, and not the further oxidation into an aldehyde. In yeast and heterologous plant hosts, the C-12 position of drimendiol is therefore likely to be further oxidized by endogenous enzymes into an aldehyde and subsequently converted to cinnamolide, presumably by spontaneous hemiacetal formation with the C-11 hydroxyl group followed by oxidation. Purified cinnamolide was confirmed by NMR and shown to be deterrent with an effective deterrent dose (ED50 ) of about 200-400 µg g-1 fresh weight against both whiteflies and aphids. The putative additional physiological and biochemical requirements for polygodial biosynthesis and stable storage in plant tissues are discussed.

Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production.

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a ß-sesquiphellandrene synthase as it was demonstrated to be functional in producing ß-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing ß-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain's endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of ß-sesquiphellandrene by 1.25-1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

Aliphatic aldehyde reductase activity related to the formation of volatile alcohols in Vietnamese coriander leaves.

Vietnamese coriander (Persicaria odorata Lour.) belongs to a group known as cilantro mimics with the 'cilantro' flavor, in which C10 and C12 aldehydes and alcohols have been found as the potent odor compounds. Their composition isolated by different extraction methods varied. The enzyme activity was assayed, and the reductase acting on some aliphatic aldehydes with NADH/NADPH as a coenzyme was found in a crude enzymatic system of fresh leaves. The maximum activity was observed at pH 8.0 in Na-phosphate and at pH 8.5 to 9.0 in a glycine-NaOH buffer, using heptanal as a substrate. The activated reductase that caused the alcohol generation to increase after a time was inhibited by p-hydroxymercuribenzoate. Our results, which are the first to be reported on Vietnamese coriander leaves, reveal the presence of aliphatic aldehyde dehydrogenase, which is responsible for acid formation, and elucidate the strong activity of the aliphatic aldehyde reductase.

New lipoxygenase-inhibiting constituents from Calligonum polygonoides.

Calligonolides A (1) and B (2), two new butanolides, and a new steroidal ester, 3, have been isolated from the whole plant of Calligonum polygonoides, together with four known compounds, tetracosan-4-olide, beta-sitosterol and its glucoside, and ursolic acid. Their structures were elucidated by spectroscopic and mass-spectrometric studies. Compounds 1-3 showed moderate inhibitory potential against lipoxygenase from soybean.

Tissue and intracellular localization of indican and the purification and characterization of indican synthase from indigo plants.

Indican (indoxyl beta-D-glucoside) was found to accumulate only in green leaves of the indigo plant, and not in any other tissues. Comparisons of the indican content of protoplasts and vacuoles showed that indican was stored only in the vacuole of the cell. Indican content appeared and increased with the appearance and growth of leaves. In mature plants, the younger leaves contained larger amounts of indican than the older ones. Cell extracts of young leaves of indigo plant catalyzed the synthesis of indican from UDP-glucose and indoxyl. Indican synthase was extracted and purified from young leaves. The enzyme was separated into two fractions by anion-exchange chromatography. The enzyme in the fraction which was eluted by 0.1 M NaCl had a molecular weight of 53,000 by SDS-PAGE. Optimum pH of the enzyme was at about 10.0, indicating that the enzyme is likely localized in a different intracellular compartment from that of indican storage. The enzyme showed normal Michaelis-Menten kinetics and a K(m) value of 0.13 mM for UDP-glucose.

beta-Glucosidase in the indigo plant: intracellular localization and tissue specific expression in leaves.

beta-Glucosidase of indigo plant (Polygonum tinctorium) has a high substrate specificity for indican (indoxyl beta-D-glucoside). To examine the localization of this beta-glucosidase, we fractionated the cells of the leaves and analysed them immunocytochemically. Immunoelectron micrographs with specific antibodies against the beta-glucosidase clearly showed that the beta-glucosidase was localized in the stroma of the chloroplasts in mesophyll cells, but not in the thylakoid membrane. Chloroplasts were isolated from the crude homogenate of the fresh leaves by Percoll density gradient centrifugation and then subjected to suborganellar fractionation. beta-Glucosidase activity was specifically detected in the stromal fraction, but not in the thylakoid membrane. This was also supported by the result of an immunoblot of the fraction with anti-beta-glucosidase antibodies. The beta-glucosidase was immunocytochemically localized in the chloroplasts of mesophyll cells, but not in any chloroplasts in marginal cells of the vascular bundle or epidermal cells; ribulose 1,5-bisphosphate carboxylase (Rubisco), a typical stromal protein, was observed in all chloroplasts in these cells. These results suggest that beta-glucosidase is tissue specific in its expression in the leaves of the indigo plant.