Correction: Pterin-based small molecule inhibitor capable of binding to the secondary pocket in the active site of ricin-toxin A chain.

[This corrects the article DOI: 10.1371/journal.pone.0277770.].

Mycorrhizal specificity differences in epiphytic habitat: three epiphytic orchids harbor distinct ecological and physiological specificity.

Orchidaceae has diversified in tree canopies and accounts for 68% of vascular epiphytes. Differences in mycorrhizal communities among epiphytic orchids can reduce species competition for mycorrhizal fungi and contribute to niche partitioning, which may be a crucial driver of the unusual species diversification among orchids. Mycorrhizal specificity-the range of fungi allowing mycorrhizal partnerships-was evaluated by assessment of mycorrhizal communities in the field (ecological specificity) and symbiotic cultures in the laboratory (physiological specificity) for three epiphytic orchids inhabiting Japan. Mycorrhizal communities were assessed with co-existing individuals growing within 10 cm of each other, revealing that ecological specificity varied widely among the three species, ranging from dominance by a single Ceratobasidiaceae fungus to diverse mycobionts across the Ceratobasidiaceae and Tulasnellaceae. In vitro seed germination tests revealed clear differences in physiological specificity among the three orchids, and that the primary mycorrhizal partners contributed to seed germination. In vitro compatibility ranges of three orchids strongly reflect the mycorrhizal community composition of wild populations. This suggests that differences in in situ mycorrhizal communities are not strongly driven by environmental factors, but are primarily due to physiological differences among orchid species. This study shows that the symbiotic strategy among the epiphytic orchid species varies from specialized to generalized association, which may contribute to biotic niche partitioning.

Crystal structure of ricin toxin A chain complexed with a highly potent pterin-based small-molecular inhibitor.

Ricin toxin A chain (RTA), from Ricinus communis, is a deadly protein that inactivates ribosomes by degrading an adenine residue at position 4324 in 28S rRNA. Recently, we have demonstrated that pterin-7-carboxamides with peptide pendants were potent RTA inhibitors. Among these, N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY) is the most potent RTA inhibitor as a small organic molecule. However, despite this fascinating inhibitory activity, the mode of interaction of 7PCGY with RTA remains elusive. This study aimed to elucidate the factors responsible for the high RTA inhibitory activity of 7PCGY based on X-ray crystallographic analysis. Herein, we report the successfully resolved X-ray crystal structure of 7PCGY/RTA complexes, revealing that the interaction between the phenolic hydroxy group in 7PCGY and Asn78 of RTA through a hydrogen bonding and the conformational change of Tyr80 and Asn122 are responsible for the high RTA inhibitory activity of 7PCGY.

Structure of pyridoxal 5'-phosphate-bound D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase (DTA) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes the reversible aldol reaction of glycine with a corresponding aldehyde to yield the D-form ß-hydroxy-α-amino acid. This study produced and investigated the crystal structure of DTA from Chlamydomonas reinhardtii (CrDTA) at 1.85 Šresolution. To our knowledge, this is the first report on the crystal structure of eukaryotic DTA. Compared with the structure of bacterial DTA, CrDTA has a similar arrangement of active-site residues. On the other hand, we speculated that some non-conserved residues alter the affinity for substrates and inhibitors. The structure of CrDTA could provide insights into the structural framework for structure-guided protein engineering studies to modify reaction selectivity.

Pterin-based small molecule inhibitor capable of binding to the secondary pocket in the active site of ricin-toxin A chain.

The Ricin toxin A chain (RTA), which depurinates an adenine base at a specific region of the ribosome leading to death, has two adjacent specificity pockets in its active site. Based on this structural information, many attempts have been made to develop small-molecule RTA inhibitors that simultaneously block the two pockets. However, no attempt has been successful. In the present study, we synthesized pterin-7-carboxamides with tripeptide pendants and found that one of them interacts with both pockets simultaneously to exhibit good RTA inhibitory activity. X-ray crystallographic analysis of the RTA crystal with the new inhibitor revealed that the conformational change of Tyr80 is an important factor that allows the inhibitors to plug the two pockets simultaneously.

Conformational change in ricin toxin A-Chain: A critical factor for inhibitor binding to the secondary pocket.

Ricin toxin A-chain (RTA), a toxic protein from Ricinus communis, inactivates ribosomes to induce toxicity. The active site of RTA consists of two binding pockets. Many studies have focused on developing RTA inhibitors that can simultaneously bind to these critical pockets; however, almost all the inhibitors developed so far interact with only one pocket. In the present study, we discovered that pterin-7-carboxamides with aromatic l-amino acid pendants interacted with the active site of the enzyme in a 2-to-1 mode, where one inhibitor molecule bound to the primary pocket and the second one entered the secondary pocket in the active site of RTA. X-ray crystallographic analysis of inhibitor/RTA complexes revealed that the conformational changes of Tyr80 and Asn122 in RTA were critical for triggering the entry of inhibitor molecules into the secondary pocket of the RTA active site.

Conformational changes in the catalytic region are responsible for heat-induced activation of hyperthermophilic homoserine dehydrogenase.

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.

The 5-Ketofructose Reductase of Gluconobacter sp. Strain CHM43 Is a Novel Class in the Shikimate Dehydrogenase Family.

Gluconobacter sp. strain CHM43 oxidizes mannitol to fructose and then oxidizes fructose to 5-keto-d-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here, we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP+-dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP+-dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the Km value for 5KF but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the Km value for 5KF, suggesting a catalytic mechanism similar to that of SDH. With these data taken together, we suggest that KFR is a new member of the SDH family. IMPORTANCE A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose, a potential low-calorie sweetener, at a high yield. Here, we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation, and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small subgroup of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved, and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.

X-ray structure analysis of a unique D-amino-acid oxidase from the thermophilic fungus Rasamsonia emersonii strain YA.

D-Amino-acid oxidases (DAAOs) catalyze the oxidative deamination of neutral and basic D-amino acids. The DAAO from the thermophilic fungus Rasamsonia emersonii strain YA (ReDAAO) has a high thermal stability and a unique broad substrate specificity that includes the acidic D-amino acid D-Glu as well as various neutral and basic D-amino acids. In this study, ReDAAO was crystallized by the hanging-drop vapor-diffusion method and its crystal structure was determined at a resolution of 2.00 Å. The crystal structure of the enzyme revealed that unlike other DAAOs, ReDAAO forms a homotetramer and contains an intramolecular disulfide bond (Cys230-Cys285), suggesting that this disulfide bond is involved in the higher thermal stability of ReDAAO. Moreover, the structure of the active site and its vicinity in ReDAAO indicates that Arg97, Lys99, Lys114 and Ser231 are candidates for recognizing the side chain of D-Glu.

Mechanism of eukaryotic serine racemase-catalyzed serine dehydration.

Eukaryotic serine racemase (SR) is a pyridoxal 5'-phosphate enzyme belonging to the Fold-type II group, which catalyzes serine racemization and is responsible for the synthesis of D-Ser, a co-agonist of the N-methyl-d-aspartate receptor. In addition to racemization, SR catalyzes the dehydration of D- and L-Ser to pyruvate and ammonia. The bifuctionality of SR is thought to be important for D-Ser homeostasis. SR catalyzes the racemization of D- and L-Ser with almost the same efficiency. In contrast, the rate of L-Ser dehydration catalyzed by SR is much higher than that of D-Ser dehydration. This has caused the argument that SR does not catalyze the direct D-Ser dehydration and that D-Ser is first converted to L-Ser, then dehydrated. In this study, we investigated the substrate and solvent isotope effect of dehydration of D- and L-Ser catalyzed by SR from Dictyostelium discoideum (DdSR) and demonstrated that the enzyme catalyzes direct D-Ser dehydration. Kinetic studies of dehydration of four Thr isomers catalyzed by D. discoideum and mouse SRs suggest that SR discriminates the substrate configuration at C3 but not at C2. This is probably the reason for the difference in efficiency between L- and D-Ser dehydration catalyzed by SR.

Synthesis and Aldose Reductase Inhibitory Activity of Botryllazine A Derivatives.

Aldose reductase (AR) is associated with the onset of diabetic complications. Botryllazine A and its analogues were synthesized and evaluated for human AR inhibitory activity. Analogues possessing aromatic bicyclic systems at the C5 position of the central pyrazine ring exhibited superior AR inhibiting activity relative to the parent botryllazine A. In addition, the benzoyl groups at positions C2 and C3 of the pyrazine ring were dispensable for this improved inhibitory activity. Conversely, a benzoyl group-containing phenolic hydroxyl groups-at either position C2 or C3 of the pyrazine ring was essential for attainment of high inhibitory activity approaching that of sorbinil (a highly effective AR inhibitor).

Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex.

Homoserine dehydrogenase (EC 1.1.1.3, HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate. Here, HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) was found to be inhibited by cysteine, which acted as a competitive inhibitor of homoserine with a Ki of 11 µM and uncompetitive an inhibitor of NAD and NADP with Ki's of 0.55 and 1.2 mM, respectively. Initial velocity and product (NADH) inhibition analyses of homoserine oxidation indicated that StHSD first binds NAD and then homoserine through a sequentially ordered mechanism. This suggests that feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Structural analysis of StHSD complexed with cysteine and NAD revealed that cysteine situates within the homoserine binding site. The distance between the sulfur atom of cysteine and the C4 atom of the nicotinamide ring was approximately 1.9 Å, close enough to form a covalent bond. The UV absorption-difference spectrum of StHSD with and without cysteine in the presence of NAD, exhibited a peak at 325 nm, which also suggests formation of a covalent bond between cysteine and the nicotinamide ring.

Crystal structure of a pyridoxal 5'-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii.

Aspartate racemase (AspR) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Šresolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the ß-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

Crystallization and X-ray analysis of D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase from the green alga Chlamydomonas reinhardtii (CrDTA) catalyzes the interconversion of several ß-hydroxy-D-amino acids (e.g. D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Šresolution. Analysis of the diffraction pattern showed that the crystal belonged to space group P1, with unit-cell parameters a = 64.79, b = 74.10, c = 89.94 Å, α = 77.07, ß = 69.34, γ = 71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å3 Da-1 and the solvent content was 41.9%.

Possible role of a histidine residue in the substrate specificity of yeast d-aspartate oxidase.

D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids, whereas neutral and basic D-amino acids are substrates of D-amino acid oxidase (DAO). DDO of the yeast Cryptococcus humicola (ChDDO) has much higher substrate specificity to D-aspartate, but the structural features that confer this specificity have not been elucidated. A three-dimensional model of ChDDO suggested that a histidine residue (His56) in the active site might be involved in the unique substrate specificity, possibly through the interaction with the substrate side chain in the active site. His56 mutants with several different amino acid residues (H56A, H56D, H56F, H56K and H56N) exhibited no significant activity toward acidic D-amino acids, but H56A and H56N mutants gained the ability to utilize neutral D-amino acids as substrates, such as D-methionine, D-phenylalanine and D-glutamine, showing the conversion of ChDDO to DAO by these mutations. This conversion was also demonstrated by the sensitivity of these mutants to competitive inhibitors of DAO. These results and kinetic properties of the mutants show that His56 is involved in the substrate specificity of ChDDO and possibly plays a role in the higher substrate specificity toward D-aspartate.

Domain characterization of Bacillus subtilis GabR, a pyridoxal 5'-phosphate-dependent transcriptional regulator.

Bacillus subtilis GabR is a transcriptional regulator consisting of a helix-turn-helix N-terminal DNA-binding domain, a pyridoxal 5'-phosphate (PLP)-binding C-terminal domain that has a structure homologous to aminotransferases, and a linker of 29 amino acid residues. In the presence of γ-aminobutyrate (GABA), GabR activates the transcription of gabT and gabD, which encode GABA aminotransferase and succinate semialdehyde dehydrogenase, respectively. We expressed N-terminal and C-terminal domain fragments (named N'-GabR and C'-GabR) in Escherichia coli cells, and obtained N'-GabR as a soluble monomer and C'-GabR as a soluble dimer. Spectroscopic studies suggested that C'-GabR contains PLP and binds to d-Ala, ß-Ala, d-Asn and d-Gln, as well as GABA, although the intact GabR binds only to GABA. N'-GabR does not bind to the DNA fragment containing the GabR-binding sequence regardless of the presence or absence of C'-GabR. A fusion protein consisting of N'-GabR and 2-aminoadipate aminotransferase of Thermus thermophilus bound to the DNA fragment. These results suggested that each domain of GabR could be an independent folding unit. The C-terminal domain provides the N-terminal domain with DNA-binding ability via dimerization. The N-terminal domain controls the ligand specificity of the C-terminal domain. Connection by the linker is indispensable for the mutual interaction of the domains.

Role of the aminotransferase domain in Bacillus subtilis GabR, a pyridoxal 5'-phosphate-dependent transcriptional regulator.

MocR/GabR family proteins are widely distributed prokaryotic transcriptional regulators containing pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B6. The Bacillus subtilis GabR, probably the most extensively studied MocR/GabR family protein, consists of an N-terminal DNA-binding domain and a PLP-binding C-terminal domain that has a structure homologous to aminotransferases. GabR suppresses transcription of gabR and activates transcription of gabT and gabD, which encode γ-aminobutyrate (GΑΒΑ) aminotransferase and succinate semialdehyde dehydrogenase, respectively, in the presence of PLP and GABA. In this study, we examined the mechanism underlying GabR-mediated gabTD transcription with spectroscopic, crystallographic and thermodynamic studies, focusing on the function of the aminotransferase domain. Spectroscopic studies revealed that GABA forms an external aldimine with the PLP in the aminotransferase domain. Isothermal calorimetry demonstrated that two GabR molecules bind to the 51-bp DNA fragment that contains the GabR-binding region. GABA minimally affected ΔG(binding) upon binding of GabR to the DNA fragment but greatly affected the contributions of ΔH and ΔS to ΔG(binding). GABA forms an external aldimine with PLP and causes a conformational change in the aminotransferase domain, and this change likely rearranges GabR binding to the promoter and thus activates gabTD transcription.

Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction.

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.

Catalytic mechanism of serine racemase from Dictyostelium discoideum.

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of D- and L-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to L-serine dehydrase; S81A showed no racemase activity and had significantly reduced D-serine dehydrase activity, but it completely retained its L-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove D-serine dehydration by abstracting the α-hydrogen in D-serine. Our data suggest that the abstraction and addition of α-hydrogen to L- and D-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.

Metal ion dependency of serine racemase from Dictyostelium discoideum.

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2 µM and 2.2 mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.