Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases

Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.     

Metabolic differences between gymnosperms and angiosperms in the formation of syringyl lignin

Sliced xylem tissue from shoots of both poplar and cherry reduces ferulic and sinapic acids to the corresponding aldehydes and alcohols, while tissue from gymnosperms such as Japanese red pine and ginkgo can reduce only ferulic acid. In young, less differentiated, xylem tissue and callus tissue of angiosperms the ability to reduce sinapic acid is markedly lower than that of the fully differentiated xylem.Both gymnosperm and angiosperm tissues reduced coniferyl and sinapyl aldehydes to the corresponding alcohols and, further, the peroxidases from both classes gave similar dehydrogenation polymers from a mixture of coniferyl and sinapyl alcohols. In agreement with these findings, sinapyl aldehyde and sinapyl alcohol, when fed to living plants and tissue cultures of gymnosperms, were shown to be readily converted to syringyl lignin which was not originally present.     

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.     

Towards the specification of consecutive steps in macromolecular lignin assembly

When Pinus taeda cell suspension cultures are exposed to 8% sucrose solution, the cells undergo significant intracellular disruption, irregular wall thickening/lignification with concomitant formation of an 'extracellular lignin precipitate. However, addition of potassium iodide (KI), an H202 scavenger, inhibits this lignification response, while the ability to synthesize the monolignols, p-coumaryl and coniferyl alcohols, is retained. Lignin synthesis (i.e. polymerization) is thus temporarily correlated with H202 generation, strongly implying a regulatory role for the latter. Time course analyses of extracellular metabolites leading up to polymer formation reveal that coniferyl alcohol, but not p-coumaryl alcohol, undergoes substantial coupling reactions to give various lignans. Of these, the metabolites, dihydrodehydrodiconiferyl alcohol, shonanin (divanillyl tetrahydrofuran) and its apparent aryl tetralin derivative, cannot be explained simply on the basis of phenolic coupling. It is proposed that these moieties are the precursors of so-called reduced substructures in the lignin macromolecule. This adds a new perspective to the lignin assembly mechanism.     

Characterization of Two Isozymes of Coniferyl Alcohol Dehydrogenase from Streptomyces sp. NL15-2K

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.     

Coniferyl alcohol metabolism in conifers -- I. Glucosidic turnover of cinnamyl aldehydes by UDPG: coniferyl alcohol glucosyltransferase from pine cambium.

UDPG: coniferyl alcohol glucosyltransferase (CAGT; EC 2.4.1.111) isolated from cambial tissues of Pinus strobus was able to convert cinnamyl aldehydes as well as dihydroconiferyl alcohol into their corresponding 4-O-beta-D-glucosides in vitro. Cinnamyl aldehydes were glucosylated with comparable efficiency to coniferyl alcohol, the physiological substrate for CAGT. Seasonal patterns of CAGT activity for aldehydes were similar to those of coniferyl alcohol. Formation of cinnamyl aldehyde and additional monolignol glucosides indicates that precursor flux and availability for lignification is likely greater than previously recognized.     

Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP⁺ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.     

Chemical syntheses of caffeoyl and 5-OH coniferyl aldehydes and alcohols and determination of lignin O-methyltransferase activities in dicot and monocot species.

To investigate the substrate preferences of O-methyltransferases in the monolignol biosynthetic pathways, caffeoyl and 5-hydroxy coniferyl aldehydes were synthesized by a new procedure involving a Wittig reaction with the corresponding hydroxybenzaldehydes. The same procedure can also be used to synthesize caffeoyl and 5-hydroxyconiferyl alcohols. Relative O-methyltransferase activities against these substrates were determined using crude extracts and recombinant caffeic acid O-methyltransferase from alfalfa (Medicago sativa), and crude extracts from the model legume Medicago truncatula, tobacco, wheat and tall fescue. Extracts from all these species catalyzed methylation of the various monolignol aldehydes and alcohols more effectively than the corresponding hydroxycinnamic acids.     

Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.     

Fermentation of 1,2-Propanediol and 1,2-Ethanediol by Some Genera of Enterobacteriaceae, Involving Coenzyme B12-Dependent Diol Dehydratase

Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2-diols, to acids and alcohols. The presence of activities of coenzyme B(12)-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera of Enterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B(12)-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B(12) was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2-propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.     

The role of alcohols in pheromone biosynthesis by two noctuid moths that use acetate pheromone components

Primary alcohols varying in chain length from C₁₃ to C₁₆, and in number, position, and geometric configuration of double bonds, were applied in dimethyl sulfoxide to the surface of the female sex pheromone glands of Heliothis subflexa (Gn.) and Hydraecia micacea (Esper). Capillary gas chromatographic analysis of extracts of the treated glands indicated that the alcohols were converted to the corresponding aldehydes by H. subflexa females and to the acetates by H. micacea females. Conversions of the alcohols showed no preferences for molecular weight, number, position, or geometry of the double bonds in either species. Application of the acetates of the primary alcohols to the gland surface of H. subflexa females resulted in the production of both the corresponding alcohols and aldehydes, while neither alcohols nor aldheydes were produced when acetates were applied to the glands of H. micacea. In addition, application of the acetates to the gland surface of Heliothis virescens (F.) resulted in the production of both the corresponding alcohols and aldehydes. However, no evidence was found to indicate that acetates are ever produced by the pheromone gland of females of H. virescens.     

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Alcohol dehydrogenase (E. C. 1.1.1.1) from Thermoanaerobium brockii at 25 degrees C and at 65 degrees C is more active with secondary than primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (V(m)) for secondary alcohols at 65 degrees C are 10 to 100 times higher than those at 25 degrees C, whereas the K(m) values are more comparable.At both 25 degrees C and 65 degrees C the substrate analogue 1,1,1,3,3,3-hexafluoro-2-propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order.At 25 degrees C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept-linear, slope-parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone.At 65 degrees C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration. Experiments in which NADP was the variable substrate and cyclopentanol as the constant-variable substrate over a wide range of concentrations gave double reciprocal plots in which the intercepts showed substrate activation and the slopes showed substrate inhibition. These results indicate that the secondary alcohols bind to the enzyme-NADPH complex at high concentrations and that the resulting ternary complex dissociates NADPH faster than the enzyme-NADPH complex. (c) 1993 John Wiley & Sons, Inc.     

A sensitive high-performance liquid chromatographic-fluorometric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to greater than or equal to 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 micrograms). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 nM methotrexate. The Km of the enzyme for NADPH was found to be 7.5 microM, while the Km for 7,8-dihydrobiopterin was 88 microM. Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.     

Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants

The effects of cinnamyl alcohol dehydrogenase (CAD, EC.1.1.1.195) down-regulation on lignin profiles of plants were analysed in four selected transgenic lines of tobacco (Nicotiana tabacum L. cv. Samsun) exhibiting different levels of CAD activity (8–56% of the control). A significant decrease in thioacidolysis yields (i.e. yield of β-O-4 linked monomers) and in the ratio of syringyl to guaiacyl monomers (S/G) was observed for three transgenic lines and the most drastic reduction (up to 50%) was correlated with the lowest level of CAD activity. Higher lignin extractability by mild alkali treatment was confirmed, and, in addition to a tenfold increase in C₆-C₁ aldehydes, coniferyl aldehyde was detected by high-performance liquid chromatography in the alkali extracts from the xylem of transgenic plants. In-situ polymerisation of cinnamyl aldehydes in stem sections of untransformed tobacco gave a xylem cell wall coloration strikingly similar to the reddish-brown coloration of the xylem of antisense CAD-down-regulated plants. Overall, these data provide new arguments for the involvement of polymerised cinnamyl aldehydes in the formation of the red-coloured xylem of CAD-down-regulated plants. Received: 24 January 1997 / Accepted: 14 May 1997     

Identification and characterisation of Arabidopsis glycosyltransferases capable of glucosylating coniferyl aldehyde and sinapyl aldehyde

This study describes the substrate recognition profile of UGT72E1, an UDP-glucose:glycosyltransferase of Arabidopsis thaliana that is the third member of a branch of glycosyltransferases, capable of conjugating lignin monomers and related metabolites. The data show that UGT72E1, in contrast to the two closely related UGTs 72E2 and 72E3, is specific for sinapyl and coniferyl aldehydes. The biochemical properties of UGT72E1 are characterised, and are compared with that of UGT72E2, which is capable of glycosylating the aldehydes as well as coniferyl and sinapyl alcohols.     

Formation of coniferyl alcohol from ferulic acid by the white rot fungus Trametes

The white rot fungus, Trametes sp., was cultivated in a medium containing ferulic acid, glucose and ethanol under aerobic conditions in submerged culture. The ferulic acid was transformed into coniferyl alcohol, coniferylaldehyde, dihydroconiferyl alcohol, vanillic acid, vanillyl alcohol, 2-methoxyhydroquinone and 2-methoxyquinone during 48–120 hr of cultivation. The amount of coniferyl alcohol in the culture reached a maximum after 90 hr with ca 40% of the initial amount of ferulic acid. Cinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, p -coumaric acid and sinapic acid were also transformed into the corresponding alcohols, benzoic acids and benzyl alcohols in the fungus culture.     

Pyridine nucleotide specificity of barley nitrate reductase

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH.