Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.     

Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco

Plants contain a large number of proteins homologous to isoflavone reductase, an NADPH-dependent reductase involved in the biosynthesis of isoflavonoid phytoalexins in legumes. Although some are bona fide isoflavone reductases, others may catalyze distinct reductase reactions. Two tobacco genes, TP7 and A622, encoding isoflavone reductase-like proteins, had been previously identified from their unique expression patterns, but their functions were not known. We show here that TP7 is a tobacco phenylcoumaran benzylic ether reductase involved in lignan biosynthesis, but that A622 is not. To gain insight into the possible function of A622, we analyzed in detail the expression patterns of the A622 gene by RNA and protein blots, immunohistochemistry, and its promoter expression in transgenic Nicotiana sylvestris roots. The A622 expression patterns were qualitatively similar to those of putrescine N-methyltransferase, the first enzyme in nicotine biosynthesis, suggesting that A622 may function in the metabolism of nicotine or related alkaloids.     

Phenylcoumaran benzylic ether and isoflavonoid reductases are a new class of cross-reactive allergens in birch pollen, fruits and vegetables.

We investigated the biochemical function of the birch pollen allergen Bet v 6 and its role in the IgE-cross-reactivity between birch pollen and plant foods, and characterized Pyr c 5, a Bet v 6-related food allergen, from pear; the proteins were expressed as His-Tag fusion proteins in Eschershia coli and purified by Ni-chelate affinity chromatography under native conditions. Nonfusion proteins were obtained by factor Xa protease treatment. The highest degree of amino-acid sequence identity of Pyr c 5 and Bet v 6 was found with a plant protein related to a defense mechanism, which we have named phenylcoumaran benzylic ether reductase (PCBER) based on its ability to catalyze the NADPH-dependent reduction of 8-5' linked lignans such as dehydrodiconiferyl alcohol to give isodihydrodehydrodiconiferyl alcohol. Enzymatic assays with recombinant Pyr c 5 and Bet v 6 showed PCBER catalytic activity for both recombinant allergens. Both Pyr c 5 and Bet v 6 allergens had similar IgE binding characteristics in immunoblotting and enzyme allergosorbent tests (EAST), and bound IgE from 10 sera of birch-pollen-allergic patients including six pear-allergic subjects. EAST inhibition experiments with Pyr c 5 as the solid phase antigen suggested that homologous allergens may be present in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini (courgette), and carrot. In extracts of pear, apple, orange, and persimmon, the presence of proteins of approximately 30-35 kDa containing Bet v 6 cross-reactive epitopes was demonstrated with two Bet v 6-specific monoclonal antibodies. Recombinant Pyr c 5 triggered a strong, dose-dependent mediator release from basophils of a pear-allergic subject, suggesting that Pyr c 5 has the potential to elicit type I allergic reactions.     

Crystal Structure and Catalytic Mechanism of Leucoanthocyanidin Reductase from Vitis vinifera

Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 3₁₀ helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.     

Hinokinin biosynthesis in Linum corymbulosum Reichenb

Due to their peculiar stereochemistry and numerous biological activities, lignans are of widespread interest. As only a few biosynthetic steps have been clarified to date, we aimed to further resolve the molecular basis of lignan biosynthesis. To this end, we first established that the biologically active lignan (−)-hinokinin could be isolated from in vitro cultures of Linum corymbulosum. Two hypothetical pathways were outlined for the biosynthesis of (−)-hinokinin. In both pathways, (+)-pinoresinol serves as the primary substrate. In the first pathway, pinoresinol is reduced via lariciresinol to secoisolariciresinol by a pinoresinol–lariciresinol reductase, and methylenedioxy bridges are formed later. In the second pathway, pinoresinol itself is the substrate for formation of the methylenedioxy bridges, resulting in consecutive production of piperitol and sesamin. To determine which of the proposed hypothetical pathways acts in vivo , we first isolated several cDNAs encoding one pinoresinol-lariciresinol reductase ( PLR-Lc1 ), two phenylcoumaran benzylic ether reductases ( PCBER-Lc1 and PCBER-Lc2 ), and two PCBER-like proteins from a cDNA library of L. corymbulosum. PLR-Lc1 was found to be enantiospecific for the conversion of (+)-pinoresinol to (−)-secoisolariciresinol, which can be further converted to give (−)-hinokinin. Hairy root lines with significantly reduced expression levels of the plr-Lc1 gene were established using RNAi technology. Hinokinin accumulation was reduced to non-detectable levels in these lines. Our results strongly indicate that PLR-Lc1 participates in (−)-hinokinin biosynthesis in L. corymbulosum by the first of the two hypothetical pathways via (−)-secoisolariciresinol.     

Enoate reductases of Clostridia. Cloning, sequencing, and expression

The enr genes specifying enoate reductases of Clostridium tyrobutyricum and Clostridium thermoaceticum were cloned and sequenced. Sequence comparison shows that enoate reductases are similar to a family of flavoproteins comprising 2,4-dienoyl-coenzyme A reductase from Escherichia coli and old yellow enzyme from yeast. The C. thermoaceticum enr gene product was expressed in recombinant Escherichia coli cells growing under anaerobic conditions. The recombinant enzyme was purified and characterized.     

Ribonucleotide reductases: the evolution of allosteric regulation.

Ribonucleotide reductases catalyze in all living organisms the production of the deoxyribonucleotides required for DNA replication and repair. Their appearance during evolution was a prerequisite for the transition from the "RNA world," where RNA sufficed for both catalysis and information transfer, to today's situation where life depends on the interplay among DNA, RNA, and protein. Three classes of ribonucleotide reductases exist today, widely differing in their primary and quaternary structures but all with a highly similar allosteric regulation of their substrate specificity. Here, I discuss the diversities between the three classes, describe their allosteric regulation, and discuss the evidence for their evolution. The appearance of oxygen on earth provided the likely driving force for enzyme diversification. From today's characteristics of the three classes, including their allosteric regulation, I propose that the anaerobic class III reductases with their iron-sulfur cluster and the requirement for S-adenosylmethionine for the generation of a glycyl protein free radical are the closest relatives to an ancestor ribonucleotide reductase.     

An historical perspective on lignan biosynthesis: Monolignol,allylphenol and hydroxycinnamic acid coupling and downstream metabolism

This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [¹³C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – secoisolariciresinol dehydrogenase. This revised version was published online in June 2006 with corrections to the Cover Date.     

The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

The methionine sulfoxide reductases: Catalysis and substrate specificities

Oxidation of Met residues in proteins leads to the formation of methionine sulfoxides (MetSO). Methionine sulfoxide reductases (Msr) are ubiquitous enzymes, which catalyze the reduction of the sulfoxide function of the oxidized methionine residues. In vivo, the role of Msrs is described as essential in protecting cells against oxidative damages and to play a role in infection of cells by pathogenic bacteria. There exist two structurally-unrelated classes of Msrs, called MsrA and MsrB, with opposite stereoselectivity towards the S and R isomers of the sulfoxide function, respectively. Both Msrs present a similar three-step catalytic mechanism. The first step, called the reductase step, leads to the formation of a sulfenic acid on the catalytic Cys with the concomitant release of Met. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the catalysis, in particular of the reductase step, and in structural specificities.     

Purification and properties of aldehyde reductases from human placenta

Aldehyde reductases (alcohol: NADP⁺-oxidoreductases, EC 1.1.1.2) I and II from human placenta have been purified to homogeneity. Aldehyde reductase I, molecular weight about 74 000, is a dimer of two nonidentical subunits of molecular weigths of about 32 500 and 39 000, whereas aldehyde erductase II is a monomer of about 32 500. Aldehyde reductase I can be dissociated into subunits under high ionic concentrations. The isoelectric pH for aldehyde reductases I and II are 5.76 and 5.20, respectively. Amino acid compositions of the two enzymes are significantly different. Placenta aldehyde reductase I can utilize glucose with a lower affinity, whereas aldehyde reductase II is not capable to reducing aldo-sugars. Similarly, aldehyde reductase I does not catalyse the reduction of glucuronate while aldehyde reductase II has a high affinity for glucuronate. Both enzymes, however, exhibit strong affinity towards various other aldehydes such as glyceraldehyde, propionaldehyde, and pyridine-3-aldehyde. The pH optima for aldehyde reductases I and II are 6.0 and 7.0, respectively. Aldehyde reductaase I can use both NADH and NADPH as cofactors, whereas aldehyde reductase II activity is dependent on NADPH only. Both enzymes are susceptible to inhibition by sulfhydryl group reagents, aldose reductase inhibitors, lithium sulfate, and sodium chloride to varying degrees.     

Isolation and characterization of indole-3-acetaldehyde reductases from Cucumis sativus.

In a continuing study of the biosynthetic pathway and regulatory mechanisms governing indole-3-acetic acid (auxin) formation, we report the isolation and initial characterization of three distinct indole-3-acetaldehyde reductases from cucumber seedlings. These enzymes catalyze the reduction of indole-3-acetaldehyde to indole-3-ethanol with the concomitant oxidation of NAD(P)H to NAD(P)+. Two of the reductases are specific for NADPH as second substrate, while the third is specific for NADH. The enzymes show a strong specificity for indoleacetaldehyde, with apparent Km values of 73mum, 130mum, and 400mum being calculated for the two NADPH-specific reductases and the NADH-specific reductase, respectively. Under no conditions of substrate concentration, incubation time, or assay method could the reverse reaction be observed. Chromatography on a calibrated Sephadex gel column led to estimated molecualr weights of 52,000 and 17,000 for the NADPH-specific reductases, while a value of 33,000 was obtained for the NADH-specific reductase. Both NADPH-specific reductases showed a pH optimum of 5.2 with a secondary optimum at 7.0, and both enzymes were activated by increasing ionic strength. The NADH-specific reductase showed a pH optimum of 7.0 with a secondary optimum at 6.1 and was slightly inhibited by increasing ionic strength.     

Nitrate reductases: Structure,functions, and effect of stress factors

Structural and functional peculiarities of four types of nitrate reductases are considered: assimilatory nitrate reductase of eukaryotes, as well as cytoplasmic assimilatory, membrane-bound respiratory, and periplasmic dissimilatory bacterial nitrate reductases. Arguments are presented showing that eukaryotic organisms are capable of nitrate dissimilation. Data concerning new classes of extremophil nitrate reductases, whose active center does not contain molybdocofactor, are summarized.     

Comparative electrochemical study of superoxide reductases

Superoxide reductases are involved in relevant biological electron transfer reactions related to protection against oxidative stress caused by reactive oxygen species. The electrochemical features of metalloproteins belonging to the three different classes of enzymes were studied by potentio-dynamic techniques (cyclic and square wave voltammetry): desulfoferrodoxin from Desulfovibrio vulgaris Hildenborough, class I superoxide reductases and neelaredoxin from Desulfovibrio gigas and Treponema pallidum, namely class II and III superoxide reductases, respectively. In addition, a small protein, designated desulforedoxin from D. gigas, which has high homology with the N-terminal domain of class I superoxide reductases, was also investigated. A comparison of the redox potentials and redox behavior of all the proteins is presented, and the results show that SOR center II is thermodynamically more stable than similar centers in different proteins, which may be related to an intramolecular electron transfer function.     

Identification of cDNAs encoding pterocarpan reductase involved in isoflavan phytoalexin biosynthesis in Lotus japonicus by EST mining

Isoflavans and pterocarpans are the major biosynthetically connected phytoalexins in legumes. A search of the expressed sequence tag library of a model legume Lotus japonicus, which produces an (-)-isoflavan, for homologs of phenylcoumaran benzylic ether reductase catalyzing the reductive cleavage of dihydrofurans, yielded seven full-length cDNAs, and the encoded proteins were analyzed in vitro. Four of them cleaved the dihydrofuran of a pterocarpan medicarpin to yield an isoflavan (-)-vestitol and were designated pterocarpan reductase (PTR). Two PTRs displayed enantiospecificity to (-)-medicarpin, representing genuine L. japonicus PTRs, while the other two lacked enantiospecificity and were presumed to be evolutionarily primitive types.     

The three-dimensional structures of peptide methionine sulfoxide reductases: current knowledge and open questions

Methionine sulfoxides are easily formed in proteins exposed to reactive oxidative species commonly present in cells. Their reduction back to methionine residues is catalyzed by peptide methionine sulfoxide reductases. Although grouped in a unique family with respect to their biological function, these enzymes are divided in two classes named MsrA and MsrB, depending on the sulfoxide enantiomer of the substrate they reduce. This specificity-based classification differentiates enzymes which display no sequence homology. Several three-dimensional structures of peptide methionine sulfoxide reductases have been determined, so that members of both classes are known to date. These crystal structures are reviewed in this paper. The folds and active sites of MsrAs and MsrBs are discussed in the light of the methionine sulfoxide reductase sequence diversity.     

Ribonucleotide reductases: Substrate specificity by allostery

Ribonucleotide reductases catalyze in all living organisms the production of deoxynucleotides from ribonucleotides. A single enzyme provides a balanced supply of the four dNTPs required for DNA replication. Three different but related classes of enzymes are known. Each class catalyzes the same chemistry using a common radical mechanism involving a thiyl radical of the enzyme but the three classes employ different mechanisms for the generation of the radical. For each class a common allosteric mechanism with ATP and dNTPs as effectors directs the substrate specificity of the enzymes ensuring the appropriate balance of the four dNTPs for DNA replication. Recent crystallographic studies of the catalytic subunits from each class in combination with allosteric effectors, with and without cognate substrates, delineated the structural changes caused by effector binding that direct the specificity of the enzymes towards reduction of the appropriate substrate.     

Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.