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At least 19 recordsLinked to original sources
PubMed2005

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism.

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.

Amino Acid Sequence↗
PubMed1999

Transcriptional activation of ydeA, which encodes a member of the major facilitator superfamily, interferes with arabinose accumulation and induction of the Escherichia coli arabinose PBAD promoter.

Induction of genes expressed from the arabinose PBAD promoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to the ydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient -35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). The ydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of ydeA has no detectable effect on cell growth. Thus, ydeA appears to be nonessential under usual laboratory growth conditions. The ydeA gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of ydeA transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.

Arabinose↗
PubMed2005

L-Arabinose 1-dehydrogenase: a novel enzyme involving in bacterial L-arabinose metabolism.

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46) catalyzing conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme involved in the first step of this L-arabinose metabolic pathway. The purified enzyme was preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had a high catalytic efficiency for both L-arabinose and D-galactose and that the L-arabinose-specific configuration at C3 and C4 is important for a preference of the substrate sugar. The N-terminal and internal amino acid sequences had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase and D-galactose 1-dehydrogenases.

Amino Acid Sequence↗
PubMed2003

Production of ethanol from L-arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway.

The fungal pathway for L-arabinose catabolism converts L-arabinose to D-xylulose 5-phosphate in five steps. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose. Only some of the genes for the corresponding enzymes were known. We have recently identified the two missing genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase and shown that overexpression of all the genes of the pathway in Saccharomyces cerevisiae enables growth on L-arabinose. Under anaerobic conditions ethanol is produced from L-arabinose, but at a very low rate. The reasons for the low rate of L-arabinose fermentation are discussed.

Anaerobiosis↗
PubMed1993

Equilibrium and transient kinetic studies of the binding of cytochalasin B to the L-arabinose-H+ symport protein of Escherichia coli. Determination of the sugar binding specificity of the L-arabinose-H+ symporter.

The kinetics of the binding of cytochalasin B to the L-arabinose-H+ symport protein of Escherichia coli have been investigated, using a strain that over-produces the symport protein in the cytoplasmic membrane. Equilibrium binding studies revealed a single set of binding sites (2.9-8.9 nmol/mg protein) with a Kd of 0.7-1.0 microM at 22 degrees C. It proved possible to follow the transient kinetics of cytochalasin B binding by measuring the changes in the fluorescence of the L-arabinose-H+ symporter upon binding the ligand, by stopped-flow fluorescence spectroscopy. The association and dissociation rate constants thus determined were confirmed by rapid filtration measurements, using [3H]cytochalasin B, yielding values of 4.5-6.5 microM-1.s-1 and 4-5 s-1, respectively, consistent with Kd values obtained by measuring equilibrium binding of [3H]cytochalasin B by dialysis at 22 degrees C. Titration of the protein fluorescence with cytochalasin B yielded a similar binding site concentration and Kd value to those obtained in equilibrium binding studies. All the measurements of binding site concentration are consistent with a stoichiometry of 1 mol cytochalasin B binding sites/mol L-arabinose-H+ symport protein. Inhibition of both the rate and equilibrium binding of cytochalasin B by sugars indicated the following order of substrate binding 5-thio-D-glucose > D-fucose > L-arabinose > 6-deoxy-6-fluoro-D-galactose > D-xylose approximately 6-deoxy-D-glucose > D-galactose > D-glucose > D-ribose. Neither D-arabinose nor L-fucose had any significant inhibitory effect upon cytochalasin B binding.

Arabinose↗
PubMed1983

Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli.

The thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli have been studied by isothermal and scanning calorimetry. The binding reaction with arabinose is characterized by an enthalpy change of -15.3 +/- 0.5 kcal mol-1 at 25 degrees C, and a large decrease in apparent heat capacity, amounting to -0.44 +/- 0.05 kcal K-1 mol-1, which is constant over the temperature range 8 to 30 degrees C. Very similar results were obtained with D-galactose. These calorimetric results have been combined with binding constants determined by equilibrium dialysis (Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227-2233) to obtain free energy and entropy changes over the range 5 to 30 degrees C, and by extrapolation to 60 degrees C. The protein undergoes reversible unfolding on being heated with an increase in enthalpy at 53.5 degrees C of 151.8 +/- 1.1 kcal mol-1 (169.2 +/- 1.2 kcal mol-1 at 59.0 degrees C) and in apparent heat capacity of 3.16 +/- 0.07 kcal K-1 mol-1. In the presence of arabinose, the unfolding enthalpy is increased to 200.7 +/- 1.8 kcal mol-1 at 59.0 degrees C, the increase being due to the enthalpy of dissociation of the ligand which amounts to 31 kcal mol-1 at the unfolding temperature. The unfolding temperature is increased by the presence of excess arabinose or galactose, an effect which is due solely to displacement by the added ligand of the unfolding-dissociation equilibrium. The thermodynamic data are discussed in connection with the detailed structural information available for this system from x-ray crystallography (Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213-13217, and references cited therein).

Arabinose↗
PubMed1977

L-Arabinose transport and the L-arabinose binding protein of Escherichia coli.

The active accumulation of L-arabinose by arabinose induced cultures of Escherichia coli is mediated by 2 independent transport mechanisms. One, specified by the gene locus araE, is membrane bound and possesses a relatively "low affinity". The other, specified in part by the genetic locus araF, contains as a functional component the L-arabinose binding protein and functions with a "high affinity" for the substrate. The L-arabinose binding protein has been purified, partially characterized, crystallized, and sequenced.

Amino Acid Sequence↗
PubMed1978

Pentose metabolism in Mycobacterium smegmatis: comparison of L-arabinose isomerases induced by L-arabinose and D-galactose.

D-Galactose, which did not serve as a growth substrate, was found to induce an L-arabinose isomerase of similar properties to the L-arabinose-induced L-arabinose isomerase. In both cases the pH profiles, pH stability, optimum temperature, heat stability, substrate specificity, metal ion requirements, mobility on polyacrylamide gel electrophoresis, and kinetic properties of the induced isomerases were identical. It appears possible that D-galactose was incorporated into the cells by an L-arabinose permease system that was alos induced by D-galactose.

Arabinose↗
PubMed1988

D-arabinose metabolism in Escherichia coli B: induction and cotransductional mapping of the L-fucose-D-arabinose pathway enzymes.

D-Arabinose is degraded by Escherichia coli B via some of the L-fucose pathway enzymes and a D-ribulokinase which is distinct from the L-fuculokinase of the L-fucose pathway. We found that L-fucose and D-arabinose acted as the apparent inducers of the enzymes needed for their degradation. These enzymes, including D-ribulokinase, appeared to be coordinately regulated, and mutants which constitutively synthesized the L-fucose enzymes also constitutively synthesized D-ribulokinase. In contrast to D-arabinose-positive mutants of E. coli K-12, in which L-fuculose-1-phosphate and D-ribulose-1-phosphate act as inducers of the L-fucose pathway, we found that these intermediates did not act as inducers in E. coli B. To further characterize the E. coli B system, some of the L-fucose-D-arabinose genes were mapped by using bacteriophage P1 transduction. A transposon Tn10 insertion near the E. coli B L-fucose regulon was used in two- and three-factor reciprocal crosses. The gene encoding D-ribulokinase, designated darK, was found to map within the L-fucose regulon, and the partial gene order was found to be Tn10-fucA-darK-fucI-fucK-thyA.

Arabinose↗
PubMed2003

Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase.

Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.

Journal Article↗
PubMed1994

Quantum mechanical model assembly study on the energetics of binding of arabinose, fucose, and galactose to L-arabinose-binding protein.

Binding energies of L-arabinose, D-fucose, and D-galactose to L-arabinose-binding protein was investigated theoretically. The calculated binding energies were composed of three contributions: 1) direct ligand-active site interaction energies calculated using static ab initio model assemblies; 2) solvation energies of the ligands; and 3) long-range electrostatic interaction energies between the ligands and the protein matrix. The calculated binding energies and the contributions of the energy components were used to analyze the experimental affinities of the ligands.

Arabinose↗
PubMed1984

Crystalline 2,3:4,5-di-O-isopropylidene-DL-arabinose diethyl dithioacetal: some reactions of acetal derivatives of arabinose.

Acetonation of the diethyl dithioacetals of D- and L-arabinose gives the corresponding 2,3:4,5-diisopropylidene acetals (2a and 2b) as oils having [alpha]D +82 and -81 degrees, respectively; in admixture, the enantiomers form a well crystallized racemate, m.p. 43-45 degrees. The initial product of acetonation is the 4,5-monoisopropylidene acetal. Demercaptalation of 2a with mercury(II) chloride-cadmium carbonate gives 2,3:4,5-di-O-isopropylidene-aldehydo-D-arabinose (5) in high yield, but the literature procedure employing mercury(II) chloride-mercury(II) oxide affords a mixture of 5 and 1,2:3,4-di-O-isopropylidene-beta-D-arabinopyranose (6). A trace of acid readily and completely converts the aldehydo derivative 5 into the cyclic diacetal 6.

Acetals↗
PubMed1999

Differences in genomic macrorestriction patterns of arabinose-positive (Burkholderia thailandensis) and arabinose-negative Burkholderia pseudomallei.

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate L-arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara-). Only the Ara isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed-field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate L-arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara-) isolates and 9 Ara- soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed-field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.

Arabinose↗
PubMed2005

A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-Amino-4-deoxy-L-arabinose. Identification and function oF UDP-4-deoxy-4-formamido-L-arabinose.

Modification of the phosphate groups of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. We previously demonstrated that the enzyme ArnA catalyzes the NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid to yield the UDP-4''-ketopentose, uridine 5'-diphospho-beta-(L-threo-pentapyranosyl-4''-ulose), which is converted by ArnB to UDP-beta-(L-Ara4N). E. coli ArnA is a bi-functional enzyme with a molecular mass of approximately 74 kDa. The oxidative decarboxylation of UDP-glucuronic acid is catalyzed by the 345-residue C-terminal domain of ArnA. The latter shows sequence similarity to enzymes that oxidize the C-4'' position of sugar nucleotides, like UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase. We now show that the 304-residue N-terminal domain catalyzes the N-10-formyltetrahydrofolate-dependent formylation of the 4''-amine of UDP-L-Ara4N, generating the novel sugar nucleotide, uridine 5'-diphospho-beta-(4-deoxy-4-formamido-L-arabinose). The N-terminal domain is highly homologous to methionyl-tRNA(f)Met formyltransferase. The structure of the formylated sugar nucleotide generated in vitro by ArnA was validated by 1H and 13C NMR spectroscopy. The two domains of ArnA were expressed independently as active proteins in E. coli. Both were required for maintenance of polymyxin resistance and L-Ara4N modification of lipid A. We conclude that N-formylation of UDP-L-Ara4N is an obligatory step in the biosynthesis of L-Ara4N-modified lipid A in polymyxin-resistant mutants. We further demonstrate that only the formylated sugar nucleotide is converted in vitro to an undecaprenyl phosphate-linked form by the enzyme ArnC. Because the L-Ara4N unit attached to lipid A is not derivatized with a formyl group, we postulate the existence of a deformylase, acting later in the pathway.

Amino Sugars↗
PubMed2003

Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-arabinose.

In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886-2896) a pathway for l-Ara4N biosynthesis that begins with the ArnA-catalyzed C-4" oxidation and C-6" decarboxylation of UDP-glucuronic acid, followed by the C-4" transamination of the product to generate the novel sugar nucleotide UDP-l-Ara4N. We now show that ArnB (PmrH) encodes the relevant aminotransferase. ArnB was overexpressed using a T7lac promoter-driven construct and shown to catalyze the reversible transfer of the amino group from glutamate to the acceptor, uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate), the intermediate that is synthesized by ArnA from UDP-glucuronic acid. A 1.7-mg sample of the putative UDP-l-Ara4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1H and 13C NMR spectroscopy. ArnB, which is a cytoplasmic protein, was purified to homogeneity from an overproducing strain of E. coli and shown to contain a pyridoxal phosphate cofactor, as judged by ultraviolet/visible spectrophotometry. The pyridoxal phosphate was converted to the pyridoxamine form in the presence of excess glutamate. A simple quantitative radiochemical assay was developed for ArnB, which can be used to assay the enzyme either in the forward or the reverse direction. The enzyme is highly selective for glutamate as the amine donor, but the equilibrium constant in the direction of UDP-l-Ara4N formation is unfavorable (approximately 0.1). ArnB is a member of a very large family of aminotransferases, but closely related ArnB orthologs are present only in those bacteria capable of synthesizing lipid A species modified with the l-Ara4N moiety.

Amino Sugars↗