D-arabinitol Dehydrogenase Research Articles

A Novel NADH-linked L-Xylulose Reductase in the L-Arabinose Catabolic Pathway of Yeast

An NADH-dependent l-xylulose reductase and the corresponding gene were identified from the yeast Ambrosiozyma monospora. The enzyme is part of the yeast pathway for l-arabinose catabolism. A fungal pathway for l-arabinose utilization has been described previously for molds. In this pathway l-arabinose is sequentially converted to l-arabinitol, l-xylulose, xylitol, and d-xylulose and enters the pentose phosphate pathway as d-xylulose 5-phosphate. In molds the reductions are NADPH-linked, and the oxidations are NAD(+)-linked. Here we show that in A. monospora the pathway is similar, i.e. it has the same two reduction and two oxidation reactions, but the reduction by l-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme. The ALX1 gene encoding the NADH-dependent l-xylulose reductase is strongly expressed during growth on l-arabinose as shown by Northern analysis. The gene was functionally overexpressed in Saccharomyces cerevisiae and the purified His-tagged protein characterized. The reversible enzyme converts l-xylulose to xylitol. It also converts d-ribulose to d-arabinitol but has no activity with l-arabinitol or adonitol, i.e. it is specific for sugar alcohols where, in a Fischer projection, the hydroxyl group of the C-2 is in the l-configuration and the hydroxyl group of C-3 is in the d-configuration. It also has no activity with C-6 sugars or sugar alcohols. The K(m) values for l-xylulose and d-ribulose are 9.6 and 4.7 mm, respectively. To our knowledge this is the first report of an NADH-linked l-xylulose reductase.

Mutations which uncouple transport and phosphorylation in the D-mannitol phosphotransferase system of Escherichia coli K-12 and Klebsiella pneumoniae 1033-5P14.

Mutants of Escherichia coli K-12 were isolated which lack the normal phosphotransferase system-dependent catabolic pathway for D-mannitol (Mtl). In some mutants the pts genes for the general proteins enzyme I and histidine protein of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems were deleted. Other mutants expressed truncated mannitol-specific enzymes II (II(Mtl)) which lacked the IIA(Mtl) or IIBA(Mtl) domain(s), and the mtlA genes originated either from E. coli K-12 or from Klebsiella pneumoniae 1033-5P14. The dalD gene from Klebsiella oxytoca M5a1 was cloned on single-copy plasmids and transformed into the strains described above. This gene encodes an NAD-dependent D-arabinitol dehydrogenase (DalD) which converts D-arabinitol into D-xylulose and also converts D-mannitol into D-fructose. The different strains were used to isolate mutations which allow efficient transport of mannitol through the nonphosphorylated II(Mtl) complexes by selecting for growth on this polyhydric alcohol. More than 40 different mutants were analyzed to determine their ability to grow on mannitol, as well as their ability to bind and transport free mannitol and, after restoration of the missing domain(s), their ability to phosphorylate mannitol. Four mutations were identified (E218A, E218V, H256P, and H256Y); all of these mutations are located in the highly conserved loop 5 of the IIC membrane-bound transporter, and two are located in its GIHE motif. These mutations were found to affect the various functions in different ways. Interestingly, in the presence of all II(Mtl) variants, whether they were in the truncated form or in the complete form, in the phosphorylated form or in the nonphosphorylated form, and in the wild-type form or in the mutated form, growth occurred on the low-affinity analogue D-arabinitol with good efficiency, while only the uncoupled mutated forms transported mannitol at a high rate.

A rapid, automated enzymatic fluorometric assay for determination of D-arabinitol in serum.

A rapid enzymatic fluorometric assay for measuring D-arabinitol in serum was developed using recombinant D-arabinitol dehydrogenase from Candida albicans (rArDH). rArDH was produced in Escherichia coli and purified by dye-ligand affinity chromatography. rArDH was highly specific for D-arabinitol, cross-reacting only with xylitol (4.9%) among all polyols tested. A Cobas Fara II centrifugal autoanalyzer (Roche) was used to measure NADH fluorometrically when rArDH and NAD were added to serum extracts, and D-arabinitol concentrations were calculated from standard curves derived from pooled human serum containing known amounts of D-arabinitol. The method was precise (mean intra-assay coefficients of variation [CVs], 0.8%, and mean interassay CVs, 1.6%) and rapid (3.5 min per assay) and showed excellent recovery of added D-arabinitol in serum (mean recovery rate, 101%). The mean and median D-arabinitol/creatinine ratios were 2.74 and 2.23 microM/mg/dl, respectively, for the 11 patients with candidemia compared to 1.14 and 1.23 microM/mg/dl, respectively, for 10 healthy controls (P < 0.01). These results confirm earlier studies showing that serum D-arabinitol measurement may help to promptly diagnose invasive candidiasis. The technique shows a significant improvement in terms of accuracy, cost, simplicity, specificity, and speed compared with gas chromatography, mass spectrometry, and earlier enzymatic assays.

A short-chain dehydrogenase gene from Pichia stipitis having D-arabinitol dehydrogenase activity.

An NAD(+)-dependent D-arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated from Pichia stipitis CBS 6054 and cloned in Saccharomyces cerevisiae. The gene was isolated by screening of a lambda-cDNA library with a zymogram technique. D-Arabinitol, xylitol, D-glucitol and galactitol are substrates for the recombinant protein. With D-arabinitol as substrate the reaction product is D-ribulose. The molecular weight of the native tetramer enzyme is 110,000 Da and the monomer is 30,000 Da. The amino acid sequence is homologous to the short-chain dehydrogenase family. It is 85.5% identical to a D-arabinitol dehydrogenase from Candida albicans. The gene in P. stipitis was induced by D-arabinitol and P. stipitis was able to grow on D-arabinitol. The physiological role of D-arabinitol metabolism is discussed.

Isolation, characterization and expression of the gene that encodes d-arabinitol dehydrogenase in candida tropicalis

The gene (ARD) that encodes NAD-dependent d-arabinitol dehydrogenase (ArDH) in the pathogenic fungus Candida tropicalis (Ct) was cloned by transforming Escherichia coli (Ec) BW31M ( araC) with a plasmid library of Ct genomic DNA and selecting for d-arabinitol-utilizing ( d-arab +) clones. Plasmid DNA from a d-arab + clone retransformed fresh Ec BW31M cells to d-arab +; these cells produced both ArDH catalytic activity and a 31-kDa protein recognized by antibodies to native Ct ArDH. The plasmid contained an 846-bp open reading frame (ORF) that encoded a deduced protein of 282 amino acids (aa) (30 748 Da). Four partial aa sequences from Ct ArDH were present in the deduced aa sequence, thus verifying that Ct ARD had been cloned. Ct ArDH was 95% identical to ArDH from Candida albicans (Ca), 85% identical to a xylitol dehydrogenase (XDH) from Pichia stipitis (Ps) and 20–25% identical to many other short-chain dehydrogenases. Ct ArDH, Ca ArDH and Ps XDH were typical short-chain dehydrogenases except that they lacked an N-terminal Gly that is conserved in other members of this family. Thus, these enzymes may represent a subclass of closely-related fungal pentitol dehydrogenases. Large amounts of recombinant ArDH (re-ArDH) were produced in Ec and purified by dye ligand affinity chromatography. The physical and catalytic properties of re-ArDH were similar to those of native Ct ArDH, and re-ArDH and native ArDH performed similarly in an automated enzymatic assay for d-arabinitol in human serum.

An automated enzymatic method for measurement of D-arabinitol, a metabolite of pathogenic Candida species

An automated enzymatic method was developed for the measurement of D-arabinitol in human serum. The assay is based on a novel, highly specific D-arabinitol dehydrogenase from Candida tropicalis. This enzyme catalyzes the oxidation of D-arabinitol to D-ribulose and the concomitant reduction of NAD+ to NADH. The NADH produced is used in a second reaction to reduce p-iodonitrotetrazolium violet (INT) to INT-formazan, which is measured spectrophotometrically. The entire reaction sequence can be performed automatically on a COBAS MIRA-S clinical chemistry analyzer (Roche Diagnostic Systems, Inc., Montclair, N.J.). Replicate analyses of human sera supplemented with D-arabinitol over a concentration range of 0 to 40 microM demonstrated that the pentitol could be measured with an accuracy of +/- 7% and a precision (standard deviation) of +/- 0.4 microM. Serum D-arabinitol measurements correlated with those determined by gas chromatography (r = 0.94). The enzymatic method is unaffected by L-arabinitol, D-mannitol, or other polyols commonly found in human serum. Any of 17 therapeutic drugs potentially present in serum did not significantly influence assay performance. Data illustrating the application of the assay in patients for possible diagnosis of invasive candidiasis and the monitoring of therapeutic intervention are presented. The automated assay described here was developed to facilitate the investigation of D-arabinitol as a serum marker for invasive Candida infections.

Identification, Purification, and Characterization of a D-Arabinitol-Specific Dehydrogenase from Candida tropicalis

A novel D-arabinitol (DA) dehydrogenase was identified and purified more than 300-fold from Candida tropicalis. The enzyme is specific for DA and catalyzes the NAD +-dependent oxidation at carbon 4 to yield D-ribulose. Purification was accomplished by a combination of protamine sulfate and ammonium sulfate precipitation and dye ligand chromatography on a reactive yellow 86 column. The apparent K m of the enzyme for DA ( [NAD +] = 2.2 mM) is 39.8 mM. The apparent K m for NAD + ( [DA] = 384 mM) is 0.12 mM. The pH-optimum for the enzymatic oxidation of DA is approximately 10. Cofactor stereospecificity studies demonstrate that the enzyme catalyzes transfer of the 4(S) hydrogen of NADH with D-ribulose as substrate. The polyol substrate specificity of the present DA dehydrogenase makes the enzyme potentially useful for the development of a simple and specific method for the measurement of DA, a metabolite of pathogenic Candida spp. which has been described as a marker for disseminated candidiasis.

Arabinitol enantiomers in cerebrospinal fluid

d-Arabinitol is a metabolite of Candida species, and its presence in serum above endogenous concentration may indicate disseminated candidiasis. The o-trifluoroacetylated derivatives of arabinitol enantiomers in cerebrospinal fluid (CSF) were separated using perpentylated cyclodextrin capillary columns and measured by selected ion monitoring using negative chemical ionization mass spectrometry. The presence of d-arabinitol was confirmed using highly specific d-arabinitol dehydrogenase. The mean d l-arabinitol ratio, 16.7 ± 4.8 (range: 8.6–22.8), in CSF of the “controls” is approximately 10-fold higher than the ratio previously found in normal serum and urine. At the same time, the mean l-arabinitol concentration, 0.13 ± 0.05 (range: 0.09–0.2), is virtually identical to that in serum. Therefore, the high d l-arabinitol ratio in CSF is attributed to d-arabinitol. Persistently high d l ratios were found in a variety of diseases (without Candida infection). The finding of d-arabinitol in CSF suggests that serum d-arabinitol may originate from the brain or the spinal cord, rather than from resident Candida species in the gut, and that the accumulation of d-arabinitol in CSF may be caused by non-consumption or, conversely, the high concentration may be maintained in order to have it readily available for metabolism in the brain.

Enantioselective measurement of fungal D-arabinitol in the sera of normal adults and patients with candidiasis.

A new method was used to measure D-arabinitol enantioselectively in the sera of 27 healthy adults and four patients with candidiasis. Arabinitol was measured by gas chromatography in serum that was treated with and without the Klebsiella pneumoniae enzyme D-arabinitol dehydrogenase, lactic dehydrogenase, NAD, and sodium pyruvate. Since enzyme treatment removed 98% of 0 to 20 micrograms of D-arabinitol per ml and none of 0 to 20 micrograms of L-arabinitol per ml from spiked sera, D-arabinitol could be determined from the difference in the treated and untreated samples. The concentrations of D- and L-arabinitol in serum from normal subjects were 0.22 +/- 0.052 and 0.16 +/- 0.055 micrograms/ml, respectively, and their D-arabinitol/creatinine and L-arabinitol/creatinine ratios were 0.024 +/- 0.0089 and 0.017 +/- 0.0053 (all means +/- standard deviations). The infected patients all had markedly elevated serum D-arabinitol levels, but their L-arabinitol levels were either normal or proportionately much lower. The excess arabinitol in the sera of individuals with candidiasis is D-arabinitol, and use of enantioselective analytical methods should result in improved ability to diagnose and estimate the severity of candidiasis.

Selection of ribitol dehydrogenase hyperproducing strains in a chemostat culture of Escherichia coli 1EA at different dilution rates

The growth of the strain Escherichia coli K12 1EA in a chemostat was limited by xylitol at dilution rates 0.05, 0.10 and 0.15 per h. Specific activities of ribitol dehydrogenase and D-arabinitol dehydrogenase were assayed to study the effect of dilution rate on the course of selection of ribitol dehydrogenase hyperproducing strains. It was found that relatively small differences in dilution rates lead to great differences in the outcome of selection experiments with respect to the level and basis of enzyme synthesis.

Enzymatic fluorometric method for the determination of d-arabinitol in serum by initial rate analysis

We describe a new, simple, fluorometric assay for d-arabinitol in serum. The method is based on oxidation of d-arabinitol by d-arabinitol dehydrogenase (EC 1.1.1.11), with the concomitant reduction of NAD. The initial rate of NAD reduction, which is proportional to the d-arabinitol content of serum, can be measured with a recording spectrofluorometer. Sensitivity, specificity, recovery and reproducibility experiments gave satisfactory results. The proposed method is suitable for clinical use, and may be helpful in the diagnosis of invasive candidiasis.

Selection of Escherichia coli K12 1EA mutants with increased synthesis of ribitol dehydrogenase.

Selection of an interspecific hybrid Escherichia coli K12 1EA in a chemostat on xylitol yielded a stable mutant synthesizing a four-fold amount of ribitol dehydrogenase (EC 1.1.1.56). Subsequent cultivation of the mutant under increased selection pressure resulted in an accumulation of a mutant with 12-fold higher level of ribitol dehydrogenase relative to the parent strain 1EA. A selection during which a UV-mutagenized population of the 1EA mutant was cultivated in a chemostat on xylitol was accompanied by monitoring the activities of ribitol dehydrogenase and D-arabinitol dehydrogenase (EC 1.1.1.11) of two adjacent catabolite operons. A several-fold increase in the activity of the two enzymes was followed by further increase in the activity of ribitol dehydrogenase and a concomitant drop in the activity of D-arabinitol dehydrogenase. The two hyperproducing strains are compared with the parent mutant as to the rate of synthesis of the two dehydrogenases and growth parameters under the conditions of batch cultivation.

Population changes in a culture of Escherichia coli K12 1EA growing in a ribitol-limited chemostat

The growth of the strain Escherichia coli K12 1EA in a chemostat was limited by ribitol as the source of carbon and energy. Specific activities of ribitol dehydrogenase and D-arabinitol dehydrogenase were assayed to measure expression of the two closely linked catabolic operons rbt and dal. Population changes occuring in a chemostat were analyzed by testing single colony isolates in batch cultures: double constitutive mutant 1EA-A was first selected while later hyperproducing strains of the type 11EA and 111EA synthesizing constitutively 4 times and 8 times more ribitol dehydrogenase, respectively, prevail in the chemostat.