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.     

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.     

Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5′) and (3′) KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.     

Flagellar hook structures of Caulobacter and Salmonella and their relationship to filament structure

Native flagellar hooks from a polarly flagellated bacterium, Caulobacter crescentus, and polyhooks from a peritrichously flagellated bacterium, Salmonella typhimurium. have been studied by densitometry of electron micrographs of negatively stained specimens, followed by computerized Fourier analysis and three-dimensional reconstruction. The two structures are remarkably similar. In both cases, the subunits are arranged along a right-handed basic helix of 2.3 nm pitch with successive subunits separated by an azimuthal angle of 64 to 65 °, and there is a pronounced system of continuous 6-start grooves and ridges on the surface of the structures. The subunit of Salmonella (M_r 42,000, versus 70,000 for Caulobacter) is somewhat thinner and yields a smaller overall hook diameter. The “bent finger” subunit shape and orientation in both cases suggests that the hook could bend readily by a sliding motion in the 11-start direction at inner radii, with the 6-start groove preventing collision at outer radii. The basic helical pitch of the Salmonella hook structure, and the number of subunits per basic helical turn (5.56) makes it highly compatible with the Salmonella flagellar filament (2.6 nm pitch. 5.51 subunits per turn); so also does the elongated shape and tilt angle of the hook and flagellin subunits in the respective structures. The two structures may therefore conjoin directly in the intact flagellum, although participation of a minor protein is not ruled out by the data.     

Structure-function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high-resolution structures

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf‐type (Nii1 and Nii3), and root‐type (Nii2 and Nii4). The high‐resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe–4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3‐M175G, Nii3‐M175E, and Nii3‐M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3‐Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild‐type (Nii3‐WT) and the Met175 variants showed that the hydrophobic side‐chain of Met175 facilitated enzyme efficiency (k_cat/K_m). The Nii4‐WT has Lys449 at the equivalent position of Gln448 in Nii3‐WT. The enzyme activity assay revealed that the turnover number (k_cat) and Michaelis constant (K_m) of Nii4‐WT were lower than those of Nii3‐WT. However, the k_cat/K_m of Nii4‐WT was about 1.4 times higher than that of Nii3‐WT. A comparison of the kinetics of the Nii3‐Q448K and Nii4‐K449Q variants revealed that the change in k_cat/K_m was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low‐affinity and Nii4 is the high‐affinity aNiR.     

Crystal structures of Enoyl-ACP reductases I (FabI) and III (FabL) from B. subtilis

Enoyl-[acyl carrier protein] (ACP) reductase (ENR) is a key enzyme in type II fatty acid synthesis that catalyzes the last step in each elongation cycle. Therefore, it has been considered as a target for antibiotics. However, recent studies indicate that some pathogens have more than one ENR; in particular, Bacillus subtilis has two ENRs, FabI and FabL. The crystal structures of the ternary complexes of BsFaBI and BsFabL are found as a homotetramer showing the same overall structure despite a sequence identity of only 24%. The positions of the catalytic dyad of Tyr-(Xaa)₆-Lys in FabL are almost identical to that of FabI, but a detailed structural analysis shows that FabL shares more structural similarities with FabG and other members of the SDR (short-chain alcohol dehydrogenase/reductase) family. The apo FabL structure shows significantly different conformations at the cofactor and the substrate-binding regions, and this resulted in a totally different tetrameric arrangement reflecting the flexibility of these regions in the absence of the cofactor and substrate/inhibitor.     

Crystal structures and thermotropic properties of alkyl alpha-D-glucopyranosides and their hydrates

Thermotropic properties and crystal structures of alkyl alpha-D-glucopyranosides and their hydrates were estimated by X-ray, DSC and thermogravimetric measurements (TGA). Monohydrates rapidly lose their crystal water several degrees below the melting point of the anhydrous glucopyranosides. The melting points of the monohydrates measured in DSC pressure cells (chain length longer than seven) are lower, and the clearing points higher than those of the anhydrous glucosides. Layer distances of smectic and crystalline phases of anhydrous compounds were established. Melting points, densities and layer distances of the crystalline anhydrous glucopyranosides display strong even-odd effects. The strong decrease of these effects in the case of the monohydrates can be elucidated by the results of X-ray crystal structure analysis.     

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.     

Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship

White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 A, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt beta-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core beta-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.     

The relationship between structure and function for the sulfite reductases

The six-electron reductions of sulfite to sulfide and nitrite to ammonia, fundamental to early and contemporary life, are catalyzed by diverse sulfite and nitrite reductases that share an unusual prosthetic assembly in their active centers, namely siroheme covalently linked to an Fe₄S₄ cluster. The recently determined crystallographic structure of the sulfite reductase hemoprotein from Escherichia coli complements extensive biochemical and spectroscopic studies in revealing structural features that are key for the catalytic mechanism and in suggesting a common symmetric structural unit for this diverse family of enzymes.     

Crystal structures and enzymatic properties of three formyltransferases from archaea: environmental adaptation and evolutionary relationship

Formyltransferase catalyzes the reversible formation of formylmethanofuran from N(5)-formyltetrahydromethanopterin and methanofuran, a reaction involved in the C1 metabolism of methanogenic and sulfate-reducing archaea. The crystal structure of the homotetrameric enzyme from Methanopyrus kandleri (growth temperature optimum 98 degrees C) has recently been solved at 1.65 A resolution. We report here the crystal structures of the formyltransferase from Methanosarcina barkeri (growth temperature optimum 37 degrees C) and from Archaeoglobus fulgidus (growth temperature optimum 83 degrees C) at 1.9 A and 2.0 A resolution, respectively. Comparison of the structures of the three enzymes revealed very similar folds. The most striking difference found was the negative surface charge, which was -32 for the M. kandleri enzyme, only -8 for the M. barkeri enzyme, and -11 for the A. fulgidus enzyme. The hydrophobic surface fraction was 50% for the M. kandleri enzyme, 56% for the M. barkeri enzyme, and 57% for the A. fulgidus enzyme. These differences most likely reflect the adaptation of the enzyme to different cytoplasmic concentrations of potassium cyclic 2,3-diphosphoglycerate, which are very high in M. kandleri (>1 M) and relatively low in M. barkeri and A. fulgidus. Formyltransferase is in a monomer/dimer/tetramer equilibrium that is dependent on the salt concentration. Only the dimers and tetramers are active, and only the tetramers are thermostable. The enzyme from M. kandleri is a tetramer, which is active and thermostable only at high concentrations of potassium phosphate (>1 M) or potassium cyclic 2,3-diphosphoglycerate. Conversely, the enzyme from M. barkeri and A. fulgidus already showed these properties, activity and stability, at much lower concentrations of these strong salting-out salts.     

Crystal structures of mannan and glucomannans

Mannan and glucomannans are related polysaccharides that are widely distributed as reserve materials of certain plants, as well as in the cell walls of some softwoods and a variety of vegetative tissues. They are often crystalline in vivo and occur as a number of crystalline allomorphs. The glucomannan from the konjac plant is also exploited commercially as a thickening and gelling agent. X-ray diffraction can be used to visualize hydrated polymer systems at atomic resolution, and the molecular and crystal structures so derived can be used to study structure-function relationships. The different crystalline allomorphs of mannan are described and compared with those of cellulose. The crystal structures of konjac mannan are described and implications for the molecular basis of its gel-forming behaviour discussed.     

Discovery of novel geranylgeranyl reductases and characterization of their substrate promiscuity

Background

Geranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo.

Results

In this work, we aimed to expand the confirmed GGR activity space by searching for novel genes that function under amenable conditions for microbial mesophilic growth in conventional hosts such as Escherichia coli or Saccharomyces cerevisiae. 31 putative GGRs were selected to test for potential reductase activity in vitro on farnesyl pyrophosphate, geranylgeranyl pyrophosphate, farnesol (FOH), and geranylgeraniol (GGOH). We report the discovery of several novel GGRs exhibiting significant activity toward various polyprenyl substrates under mild conditions (i.e., pH 7.4, T?=?37 °C), including the discovery of a novel bacterial GGR isolated from Streptomyces coelicolor. In addition, we uncover new mechanistic insights within several GGR variants, including GGR-mediated phosphatase activity toward polyprenyl pyrophosphates and the first demonstration of completely hydrogenated GGOH and FOH substrates.

Conclusion

These collective results enhance the potential for metabolic engineers to manufacture a variety of isoprenoid-based biofuels, polymers, and chemical feedstocks in common microbial hosts such as E. coli or S. cerevisiae.

     

Comparison of solution structures of dihydrofolate reductases and enzyme-ligand complexes using cross-reacting antibodies

Polyclonal antibodies against dihydrofolate reductase (DHFR) from the human lymphoblastoid cell line WIL-2/M4 were used as probes to compare the antigenic structures in solution of native DHFRs obtained from a broad range of species and their complexes with substrate, cofactor, and folate antagonist inhibitors. All these antibodies could bind to the denatured human DHFR, indicating that they were specific for the primary structure of this enzyme. Denatured chicken liver and L1210 murine leukemic DHFRs competed for all of the antibodies that bound to the human enzyme, although less effectively than the denatured human enzyme, showing the presence of similar epitopes among the vertebrate enzymes. However, both direct binding and competition experiments showed low antibody cross-reactivities with native chicken liver (8%) and murine (10%) DHFRs, suggesting differences in the disposition of similar epitopes in these enzymes. The lactobacillus casei DHFR showed a low amount (less than 2%) of cross-reactivity with the antibodies while the same antibodies did not cross-react with the Escherichia coli enzyme. DHFR from soybean seedlings competed for a large proportion (70%) of the anti-human DHFR antibodies, indicating a close similarity in the antigenic structures of plant and animal DHFRs. Binary complexes of the L. casei, avian, murine, and human DHFRs with dihydrofolate, methotrexate (MTX), trimethoprim (TMP), NADPH, and NADP+ all showed significantly lower antibody binding capacity as compared with the corresponding free enzymes. Further, these ligands inhibited antibody binding to the enzyme to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of monomeric actin bound to cytochalasin D

The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin have not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD: one obtained by soaking preformed actin crystals with CD, and the other obtained by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play equally important roles in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. An ∼ 6° rotation of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure instead of being disordered, as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.     

Crystal structures of active LytM

Lysostaphin-type enzymes are metalloendopeptidases that are present in bacteriophages and in bacteria. They share the catalytic domain, but normally contain other domains as well. The well-characterized enzymes in this group are all specific for the pentaglycine crosslinks in the cell walls of some Gram-positive bacterial species. Lysostaphin-type enzymes are synthesized as secreted preproenzymes and require proteolytic activation for maturation. Although lysostaphin, the prototypical peptidase in the group, is widely used as a tool in biotechnology and developed as an antistaphylococcal agent, the detailed structure of this enzyme is unknown. So far, only one lysostaphin-type enzyme, the Staphylococcus aureus autolysin LytM, has been crystallized in its full-length, inactive form. Here, we describe the synthesis of a convenient reporter substrate, characterize the metal and pH-dependence of an active LytM fragment, and present its crystal structure in three crystal forms at different pH values that either support or do not support activity. In all structures, we find an extended, long and narrow groove that has the active site at its bottom and is delineated on the sides by the most flexible regions of the molecule. In two cases, the groove is partially filled by a loop of a neighbouring molecule in the crystal. As the loop contains three consecutive glycine residues, this crystal packing effect supports the interpretation that the groove is the substrate-binding cleft. To characterize the substrate-binding mode more closely, a phosphinate analogue of tetraglycine was synthesized. Although tetraglycine is a substrate of the active LytM fragment, the phosphinate analogue turned out to be a very poor inhibitor. Crystals that were grown in its presence contained an L+-tartrate molecule from the crystallization buffer and not the phosphinate in the active site.     

Crystal structures of cephaibols.

The crystal structures of the peptaibol antibiotics cephaibol A, cephaibol B and cephaibol C have been determined at ca. 0.9 A resolution. All three adopt a helical conformation with a sharp bend (of about 55 degrees) at the central hydroxyproline. All isovalines were found to possess the D configuration, superposition of all four models (there are two independent molecules in the cephaibol B structure) shows that the N-terminal helix is rigid and the C-terminus is flexible. There are differences in the hydrogen bonding patterns for the three structures that crystallize in different space groups despite relatively similar unit cell dimensions, but only in the case of cephaibol C does the packing emulate the formation of a membrane channel believed to be important for their biological function.     

Crystal structures of A-DNA duplexes

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G · T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G · ^m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G · C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology. © 1997 John Wiley & Sons, Inc. Biopoly 44: 45–63, 1997