Takefumi Hattori

Possible biochemical roles of oxalic acid as a low molecular weight compound involved in brown-rot and white-rot wood decays

Accumulation of oxalic acid in low nitrogen and high nitrogen nutrient cultures of brown-rot and white-rot fungi are compared with the previously reported findings on fungal production of oxalic acid. The enzymatic formation of oxalic acid from oxaloacetate and glyoxylate in brown-rot and white-rot fungi is described in comparison with other microorganisms. Possible biochemical roles of oxalic acid are discussed in relation to brown-rot and white-rot wood decays. In the brown-rot wood decay system, oxalic acid may serve as a proton source for enzymatic and non-enzymatic hydrolysis of carbohydrates and as a metal chelator. On the other hand, in the white-rot wood decay system, oxalic acid may play multiple roles, such as an inhibitior of lignin peroidases, an electron donor for producing NADH, which may be used for reduction of lignin derived quinones, a source of formate radicals to reduce dioxygen or ferric iron to yield superoxide anion radicals and ferrous iron, respectively, and a chelator for stabilization of manganic ions for lignin degradation. Similar roles of oxalic acid in other living organisms are also briefly described.

A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris

A metabolic mechanism for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris has been proposed on the basis of biochemical analyses of glucose metabolism. There was a strong correlation between glucose consumption and oxalate production. Oxalic acid was found to accumulate in the culture fluid in about 80% of the theoretical yield or about 5-fold, on the basis of the fungal biomass harvested. The results clearly indicate that glucose was not completely oxidized to CO2 by the tricarboxylic acid (TCA) cycle but converted mainly to oxalate. The determination of the 12 enzymes concerned has revealed the occurrence of the unprecedented metabolic coupling of the TCA and glyoxylate cycles that support oxalate biosynthesis. In this metabolic system, isocitrate lyase (EC 4.1.3.1), together with oxaloacetase (EC 3.7.1.1), was found to play a pivotal role in yielding oxalate from oxaloacetate via the acetate-recycling routes. Moreover, malate dehydrogenase (EC 1.1.1.37), with an extraordinarily high activity among the enzymes tested, was shown to play an important role in generating NADH by oxidation of malate to oxaloacetate. Thus, it is proposed that the wood-rotting basidiomycete acquires biochemical energy by oxidizing glucose to oxalate.

Characterization of Arabidopsis thaliana Pinoresinol Reductase, a New Type of Enzyme Involved in Lignan Biosynthesis

A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (–)-pinoresinol to (–)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (–)-pinoresinols to (+)- and (–)-lariciresinols efficiently with comparative kcat/Km values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.

New role for glyoxylate cycle enzymes in wood-rotting basidiomycetes in relation to biosynthesis of oxalic acid

The key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), were detected in varying amounts in the mycelia of the woodrotting basidiomycetes tested, although they were grown in a glucose-rich medium. The highest specific activities of ICL (0.37 U/mg protein) and MS (0.63 U/mg protein) were measured for the brown-rot basidiomycetesLaetiporus sulphureus andFomitopsis palustris, respectively. The results indicate that the glyoxylate cycle enzymes occur in wood-rotting basidiomycetes as the seemingly “constitutive” enzymes at varying levels. The glyoxylate cycle enzymes, including malate dehydrogenase (MDH), and the oxalate-producing enzymes glyoxylate dehydrogenase (GDH) and oxaloacetase (OXA) were found to have good correlation with biosynthesis of oxalic acid and fungal growth, which was also confirmed by use of an ICL inhibitor. A new role for the glyoxylate cycle is discussed in relation to oxalic acid biosynthesis in wood-rotting basidiomycetes.

At5g54160 gene encodes Arabidopsis thaliana 5-hydroxyconiferaldehyde O-methyltransferase

The function of an Arabidopsis thaliana gene, At5g54160 annotated as a caffeic acid O-methyltransferase CAOMT gene was characterized. The recombinant enzyme of this gene (AtOMT1) catalyzed the O-methylation of phenylpropanoid and flavonoid substrates. The specificity constants (kcat/Km) for 5-hydroxyconiferaldehyde (5-HCAld) and quercetin were both 0.11 μM−1·min−1. On the other hand, lignins of At5g54160-knockout Arabidopsis mutants lacked syringyl units. In addition, we showed that the gene silencing also resulted in significant accumulation of caffeyl alcohol (CaAlc). These results strongly suggested that At5g54160 gene is involved in syringyl lignin synthesis for the methylation of both 5-hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compound(s).

Purification and characteristics of a novel cytochrome c dependent glyoxylate dehydrogenase from a wood-destroying fungus Tyromyces palustris

A new glyoxylate dehydrogenase which catalyzes dehydrogenation of glyoxylate to oxalate in the presence of cytochrome c has been purified as an electrophoretically homogeneous protein from the cell‐free extracts of a wood‐destroying basidiomycete Tyromyces palustris. The enzymatic reduction of cytochrome c was dependent on glyoxylate which was found to be the best substrate among the compounds tested. The K m value for glyoxylate was determined to be 2.7 mM at the optimal pH (8.0). The UV‐visible spectra of the enzyme in oxidized and reduced forms indicate that the enzyme belongs to a family of flavohemoproteins. The flavin nucleotide isolated from the native enzyme by heat denaturation was identified as FMN. The enzyme (M r 331 000) consists of six identical homopolymers (M r of subunit 59 000), which were found to constitute a symmetric octahedral shape by electron‐microscopic observation with a negative staining method.

Oxalate Efflux Transporter from the Brown Rot Fungus Fomitopsis palustris

ABSTRACT An oxalate-fermenting brown rot fungus, Fomitopsis palustris, secretes large amounts of oxalic acid during wood decay. Secretion of oxalic acid is indispensable for the degradation of wood cell walls, but almost nothing is known about the transport mechanism by which oxalic acid is secreted from F. palustris hyphal cells. We characterized the mechanism for oxalate transport using membrane vesicles of F. palustris. Oxalate transport in F. palustris was ATP dependent and was strongly inhibited by several inhibitors, such as valinomycin and NH4+, suggesting the presence of a secondary oxalate transporter in this fungus. We then isolated a cDNA, FpOAR (Fomitopsispalustrisoxalic acid resistance), from F. palustris by functional screening of yeast transformants with cDNAs grown on oxalic acid-containing plates. FpOAR is predicted to be a membrane protein that possesses six transmembrane domains but shows no similarity with known oxalate transporters. The yeast transformant possessing FpOAR (FpOAR-transformant) acquired resistance to oxalic acid and contained less oxalate than the control transformant. Biochemical analyses using membrane vesicles of the FpOAR-transformant showed that the oxalate transport property of FpOAR was consistent with that observed in membrane vesicles of F. palustris. The quantity of FpOAR transcripts was correlated with increasing oxalic acid accumulation in the culture medium and was induced when exogenous oxalate was added to the medium. These results strongly suggest that FpOAR plays an important role in wood decay by acting as a secondary transporter responsible for secretion of oxalate by F. palustris.

The expression of a rice secondary wall-specific cellulose synthase gene, OsCesA7, is directly regulated by a rice transcription factor, OsMYB58/63.

AbstractMain conclusionA rice MYB transcription factor, OsMYB58/63, was found to directly upregulate the expression of a rice secondary wall-specific cellulose synthase gene,cellulose synthase A7(OsCesA7); in contrast, theArabidopsisputative orthologs AtMYB58 and AtMYB63 have been shown to specifically activate lignin biosynthesis. Although indirect evidence has shown that grass plants are similar to but partially different from dicotyledonous ones in transcriptional regulation of lignocellulose biosynthesis, little is known about the differences. This study showed that a rice MYB transcription factor, OsMYB58/63, directly upregulated the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 (OsCesA7). Gene co-expression analysis showed that, in rice, OsMYB58/63 and several rice MYB genes were co-expressed with genes encoding lignocellulose biosynthetic enzymes. The expression levels of OsMYB55/61, OsMYB55/61-L, OsMYB58/63, and OsMYB42/85 were commonly found to be high in culm internodes and nodes. All four MYB transcription factors functioned as transcriptional activators in yeast cells. OsMYB58/63 most strongly transactivated the expression of OsCesA7 in rice protoplasts. Moreover, recombinant OsMYB58/63 protein was bound to two distinct cis-regulatory elements, AC-II and SMRE3, in the OsCesA7 promoter. This is in sharp contrast to the role of Arabidopsis orthologs, AtMYB58 and AtMYB63, which had been reported to specifically activate lignin biosynthesis. The promoter analysis revealed that AC elements, which are the binding sites for MYB58 and MYB63, were lacking in cellulose and xylan biosynthetic genes in Arabidopsis, but present in cellulose, xylan, and lignin biosynthetic genes in rice, implying that the difference of transcriptional regulation between rice and Arabidopsis is due to the distinct composition of promoters. Our results provide a new insight into transcriptional regulation in grass lignocellulose biosynthesis.

Cloning of a cDNA encoding a NAD‐dependent formate dehydrogenase involved in oxalic acid metabolism from the white‐rot fungus Ceriporiopsis subvermispora and its gene expression analysis

The authors have proposed previously that intracellular degradation of oxalic acid via formate to CO(2) occurs in the white-rot fungus Ceriporiopsis subvermispora. The formate degradation is catalyzed by NAD-dependent formate dehydrogenase (CsFDH). In this study, two cDNAs named CsFDH1 and CsFDH2 encoding CsFDH were cloned. Each cDNA consisting of 1077 bp encodes a mature protein composed of 358 amino acid residues. The amino acid sequences of the deduced CsFDH1 and CsFDH2 showed 99% identity to each other. The predicted molecular mass for each was 39.3 kDa, which was similar to that of CsFDH purified from the vegetative mycelia of Ceriporiopsis subvermispora (purified-CsFDH). The recombinant CsFDH1 and CsFDH2 expressed by Escherichia coli showed FDH activity with similar characteristics to the purified CsFDH. However, the amount of CsFDH1 transcript from the vegetative mycelia was 236-691 times greater than that of CsFDH2. Therefore, the results strongly suggest that CsFDH1, as compared with CsFDH2, predominantly contributes to the production of the purified CsFDH.

A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes

Abstract Biochemical roles of the representative enzymes involved in carbon metabolism of glucose were investigated in relation to the fruit-body formation of the basidiomycete Flammulina velutipes. Changes in specific activities of the enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate (GLOX) and gluconeogenesis pathways were measured at different stages of development of the fungus. The enzyme activities of malate synthase (MS) and fructose bisphosphatase (FBP) as the key enzymes for the GLOX-gluconeogenesis pathways increased in mycelia during the fruit-body formation. The activities of isocitrate dehydrogenase (IDH) for the TCA cycle and NADP-linked glutamate dehydrogenase (GLTDH (NADP)) for glutamate synthesis increased more markedly. Moreover, the mycelial mat of the cultures producing fruit bodies yielded greater enzyme activities of isocitrate lyase (ICL), MS, FBP, and IDH than that of the cultures that did not produce fruit bodies. These results suggest that the GLOX-gluconeogenesis pathways as well as the glutamate synthesis have a strong correlation with the fruit-body formation of F. velutipes.