Studies on the inhibition of gluconeogenesis by oxalate

Oxalate was shown to enter isolated rat hepatocytes and to inhibit gluconeogenesis from lactate, pyruvate, and alanine, but not from glutamine, proline, propionate or dihydroxyacetone. Oxalate apparently acts by inhibiting pyruvate carboxylase (EC 6.4.1.1). It is known to inhibit the isolated enzyme, and inhibition of gluconeogenesis was much greater in a bicarbonate-deficient medium where pyruvate carboxylase activity limits the overall rate of the pathway. A slight inhibition of gluconeogenesis from asparagine was observed, suggesting that oxalate may also inhibit gluconeogenesis at another site. Chelation of extracellular Ca²⁺ does not contribute to the inhibition of gluconeogenesis. Compared to oxalate, other Ca²⁺ chelators have little effect upon gluconeogenesis. Also, oxalate inhibits gluconeogenesis effectively both in low Ca²⁺ medium and in medium containing 2.6 mM Ca²⁺. Chelation of intracellular Ca²⁺ also appears to be of little importance, since oxalate does not block the glycogenolytic effects of epinephrine, vasopressin, and angiotensin which are thought to act via Ca²⁺ as the second messenger. The inhibition of gluconeogenesis could conceivably contribute to the toxic actions of oxalate and to the hypoglycemic action of dichloroacetate, a compound that is metabolized to oxalate. However, oxalate did not cause hypoglycemia in the suckling rat, a model in vivo system very dependent upon gluconeogenesis for maintenance of normal blood glucose levels. Thus, inhibition of gluconeogenesis is probably of little importance in oxalate toxicity and the hypoglycemic effects of dichloroacetate.     

草酸脱羧酶及其应用

草酸脱羧酶是一种含锰的酶,在白腐菌中广泛存在,少数低等真菌和细菌中也能产生。目前,至少10多种草酸脱羧酶得到了分离和纯化。该酶是一种氨基酸残基在379个左右的单体酶,一般都为酸性糖蛋白,含有2个锰离子,形成2个活性区域;表面一些氨基酸被不同程度地糖基化。晶体结构和其它一些波谱学研究解释了其空间结构和可能的电子传递机制。运用PCR技术和cDNA文库技术,越来越多的草酸脱羧酶基因被克隆。已研究的该酶基因中都含有17个左右的内含子,这些内含子在活性域位置上有比较高的保守性。一些特殊氨基酸序列的存在决定了该酶的表达形式为诱导型,菌株的基因调控序列中含有一段受草酸化合物作用的序列。该酶在一些酵母和植物等异源表达系统中有成功表达的报道。该酶的应用主要集中在以下几方面：造纸废水中的草酸盐降解;食品中的草酸降解;草酸生物检测（如,临床诊断）等。     

Polysome Isolation by Sepharose Column Chromatography

Polysomes from the mouse myeloma MOPC-21 were purified by gel filtration on Sepharose 6B, 4B and 2B columns. All three columns eliminated nearly all intracellular material smaller than 40 S subunits. In addition, passage through 4B and 2B columns substantially reduced the amount of subunits and monosomes in the preparations. Purified polysomes retained structural integrity when stored at -85°C for at least nine weeks.     

Structural Basis for Bacterial Quorum Sensing-mediated Oxalogenesis

The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (β/α)₈-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr³²² as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment.     

Oxalate production at different initial Pb²+ concentrations and the influence of oxalate during solid-state fermentation of straw with Phanerochaete chrysosporium

The production of oxalate at different initial Pb²⁺ concentrations during solid-state fermentation of straw with Phanerochaete chrysosporium was investigated. It was found that the maximal peak value of oxalate concentration (22.84 mM) was detected at the initial Pb²⁺ concentration of 200 mg kg⁻¹ dry straw, while the minimum (15.89 mM) at the concentration of 600 mg Pb²⁺ kg⁻¹ dry straw, and at moderate concentration of Pb²⁺ the capability of oxalic acid secretion was enhanced. In addition, it was also found that more oxalic acid accumulation went together with better Pb²⁺ passivation effect and higher manganese peroxidase (MnP) activity. The present findings will improve the understandings of the interactions of heavy metals with white-rot fungi and the role of oxalate in lignin degradation system, which could provide useful references for more efficient treatment of Pb-contaminated lignocellulosic waste.     

Assessing the relative bioavailability of copper to fungi degrading treated wood

Copper is ubiquitous as a biocide component in wood preservatives. Some fungi detoxify copper by immobilizing copper ions with oxalate, decreasing its physiological availability (bioavailability). Decreases in copper bioavailability may also occur during wood treatment. To date, however, copper retention in wood has been measured as overall weight-to-volume concentration without an estimate of its bioavailability and without assessment of its relative contribution to preservative efficacy. Here, we gauge the bioavailability of copper ions in treated wood by using oxalate to pre-treat wood prior to colonization by a moderately copper-tolerant fungus. Copper-treated wood was treated with a gradient of sodium oxalate concentrations, rinsed thoroughly, and exposed in soil-block jars to an isolate of Serpula himantioides. Wood treated with copper ethanolamine was extremely effective in preventing decay by S. himantioides, but toxicity could repeatedly be overcome above a threshold level of oxalate pretreatment. In agar plates, copper-treated wood stimulated oxalate production by S. himantioides, but levels were less than those needed (>10 mM) to overcome copper in soil-block jars. This capacity to overcome copper using an oxalate pretreatment was absent in commercially available samples treated with co-biocide(s). Results demonstrate a useful relative measure of copper bioavailability, with potential to be modified for specific quantification.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Isolation and characterization of a new type of aerobic,oxalic acid utilizing bacteria,and proposal of Oxalicibacterium flavum gen. nov., sp. nov

A mesophilic, aerobic oxalic acid utilizing yellow-pigmented bacterium has been isolated from litter of oxalate producing plants in the region of Izmir (Turkey). It is motile by means of 1-3 polar flagella. Optimal growth occurred between 25-30 degrees C at pH 6.9. The G+C content of DNA is 62-64 mol % (Tm). Based on its morphological and biochemical features the organism belongs to the genus Pseudomonas, but differs from all the previously described species. The taxonomic relationships among strains described as or previously tentatively assigned to the genus Pseudomonas were investigated using numerical classification, DNA base composition and DNA-DNA hybridization. 16S rDNA sequences were determined for the strain TA17. On the basis of 16S rDNA sequence comparisons, physiological and biochemical characteristics, it is proposed to classify TA17T in a new genus and species for which the name Oxalicibacterium flavum gen. nov., sp. nov. is proposed. The type strain is TA17T (= NEU98T, = LMG 21571T).     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

Characterization of histone (H1B) oxalate binding protein in experimental urolithiasis and bioinformatics approach to study its oxalate interaction

The rat kidney H1 oxalate binding protein was isolated and purified. Oxalate binds exclusively with H1B fraction of H1 histone. Oxalate binding activity is inhibited by lysine group modifiers such as 4',4'-diisothiostilbene-2,2-disulfonic acid (DIDS) and pyridoxal phosphate and reduced in presence of ATP and ADP. RNA has no effect on oxalate binding activity of H1B whereas DNA inhibits oxalate binding activity. Equilibrium dialysis method showed that H1B oxalate binding protein has two binding sites for oxalate, one with high affinity, other with low affinity. Histone H1B was modeled in silico using Modeller8v1 software tool since experimental structure is not available. In silico interaction studies predict that histone H1B-oxalate interaction take place through lysine121, lysine139, and leucine68. H1B oxalate binding protein is found to be a promoter of calcium oxalate crystal (CaOx) growth. A 10% increase in the promoting activity is observed in hyperoxaluric rat kidney H1B. Interaction of H1B oxalate binding protein with CaOx crystals favors the formation of intertwined calcium oxalate dehydrate (COD) crystals as studied by light microscopy. Intertwined COD crystals and aggregates of COD crystals were more pronounced in the presence of hyperoxalauric H1B.