Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.     

D-xylose catabolism in fusarium oxysporum

Summary The initial steps of D-xylose catabolism inFusarium oxysporum have been studied. The presence of the oxidoreductase pathway for D-xylose catabolism was demonstrated. The enzymes involved, D-xylose reductase and xylitol dehydrogenase, were found to be inducible and relatively specific for D-xylose and xylitol. D-xylose isomerase was not detected.     

Production of ethanol from wood hemicellulose hydrolyzates by a xylose-fermenting yeast mutant,Candida sp. XF 217

Summary Ethanol was produced from wood chip hemicellulose hydrolyzate by a xylose-fermenting yeast mutant, Candida sp. XF 217. The rates of D-xylose consumption and ethanol production were greater under aerobic than fermentative conditions. The slow rate of fermentation under fermentative conditions could be overcome by supplementing the broth with D-xylose isomerase (glucose isomerase). The ethanol yield, as based on the sugar consumed, was approximately 90% of the theoretical value.     

Investigation of the synthesis of xylose/glucose isomerases in sixArthrobacter sp. strains

The substrate specificity of isomerases produced by six strains ofArthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A.ureafaciens B-6 strains showed low activity toward D-ribose,Arthrobacter sp. B-5 was slightly active toward L-arabinose, andA. ureafaciens B-6 andArthrobacter sp. B-2239, toward L-rhamnose. InArthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources. The synthesis of xylose/glucose isomerase induced by D-xylose inArthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol inA. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in theArthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Uptake and catabolism of D-xylose in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 grows on D-xylose as sole carbon source with a generation time of 105 to 110 min. The following activities are induced at the indicated time after the addition of the inducer, D-xylose: D-xylulokinase (5 min), D-xylose isomerase (7 to 8 min), and D-xylose transport (10 min). All other pentoses and pentitols tested failed to induce isomerase or kinase. Synthesis of D-xylose isomerase was subject to catabolite repression, which was reversed by the addition of cyclic adenosine monophosphate. Most of the radioactive counts from D-[14C]xylose were initially accumulated in the cell in the form of D-xylose or D-xylulose. D-Xylose uptake in a mutant which was deficient in D-xylose isomerase was equal to that of the wild type. The apparent Km for D-xylose uptake was 0.41 mM. Some L-arabinose was accumulated in D-xylose-induced cells, and some D-xylose was accumulated in L-arabinose-induced cells. D-Xylitol and L-arabinose competed against C-xylose uptake, but D-arabinose, D-lyxose, and L-lyxose did not. Osmotic shock reduced the uptake of D-xylose by about 50%; by equilibrium dialysis, a D-xylose-binding protein was detected in the supernatant fluid after spheroplasts were formed from D-xylose-induced cells.     

Exploring the interaction between D-xylose isomerase and D-xylose by free energy calculation

The numerical quadrature thermodynamic integration method is used to investigate enzyme-substrate interaction of D-xylose isomerase. A screening function for the coulombic interaction is introduced into the simulation to correct the effect of finite cutoff radius for the non-bonded interaction. The binding free energy difference for D-xylose with D-xylose isomerase and its N184D mutant has been calculated, and the result 3.9 ± 1.2 kJ/mol agrees well with experimental data of 4.38 kJ/mol. In addition, the structure and dynamics of enzyme-substrate complex were simulated for mutant and wild-type enzyme, respectively. Analysis of the structures and intramolecular interactions of the complexes were found to be valuable for understanding the reaction mechanism of the enzyme D-xylose isomerase. © Wiley-Liss, Inc.     

D-Glucose Isomerase: Constitutive and Catabolite Repression-Resistant Mutants of Streptomyces phaeochromogenes

As in other Streptomyces species, the enzymatic conversion of D-glucose to D-fructose is carried out in Streptomyces phaeochromogenes NRRL B-3559 by the inducible enzyme, D-xylose keto isomerase (EC 5.3.1.5). Mutants of this microorganism were selected for their ability to grow on D-lyxose (2-epimer of D-xlose). As a result of the mutational event, the microorganism constitutively produced D-xylose isomerase. As in the parent strain, the constitutive formation of the isomerase was repressed by D-glucose. The fact that this mutant was unable to grow in low D-xylose concentrations in the presence of the D-glucose analogue, 3-O-methylglucose, permitted the isolation of D-xylose isomerase constitutive mutants which were insensitive to D-glucose repression.     

Phylogenetic Relationships between different D-Xylose Biogroups in Wild-type Salmonella typhimurium strains and a suggested Evolutionary Pathway

Sixty-two wild-type Salmonella typhimurium strains were characterized for their D-xylose enzyme activities. Strains from the xylose strong biogroup synthesized high levels of D-xylose isomerase and D-xylulokinase and transported D-xylose. Strains from the xylose weak biogroup synthesized only low levels of D-xylulokinase and low, or no, levels of D-xylose isomerase and were deficient in the ability to transport D-xylose. These findings are discussed in the light of known phylogenetic relationships among the biotypes of Salm. typhimurium.     

A quasi-Laue neutron crystallographic study of d-xylose isomerase

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.     

Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2.

Twenty-one Xyl- mutants of Salmonella typhimurium were selected: all had lost one or more of the activities for D-xylose isomerase, C-xylulokinase, or D-xylose transport. The mutants were classified into five functional groups: xylR, pleiotropic negative (12 mutants); xylA, D-xylose isomerase defective (3 mutants); xylB, D-xylulokinase defective (2 mutants); xylT, D-xylose transport defective (1 mutant); and 3 mutants with defective D-xylose isomerase and D-xylulokinase. Some nonsense mutations were identified among the xylR mutants. Two F'xyl plasmids were isolated by selection for early transfer of xyl+ by an Hfr which transfers xyl as a terminal gene; a plasmid with a mutation in the xyl genes, F'xylR1, was also isolated. Complementation tests using F'xyl plasmids indicate that expression of the xylA, xylB, and xylT genes is under the positive control of the xylR regulatory gene. Conjugation crosses and P22-mediated transduction data indicate that all the xyl mutations tested are in a cluster of genes at 78 units on the linkage map, and that the gene order is xylT--xylR--xylB--xylA--glyS--mtlA,D.     

Transcriptional analysis of catabolite repression in Clostridium acetobutylicum growing on mixtures of D-glucose and D-xylose

Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably using a catabolite repression mechanism. Accordingly during growth on a mixture of D-glucose and D-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in D-xylose degradation. The first operon (CAC1344-CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by D-xylose but was catabolite repressed by D-glucose. A second operon (CAC2610-CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a L-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by D-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.     

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.     

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.     

Pentose metabolism in Mycobacterium smegmatis: specificity of induction of pentose isomerases.

The induction of D-xylose, D-ribose, L-arabinose, and D-lyxose isomerases by various sugars was studied to determine the configuration necessary for induction. D-Xylose isomerase was only induced by D-xylose, whereas D-ribose isomerase was induced by D-ribose, L-rhamnose, and L-lyxose. L-arabinose isomerase was induced by L-arabinose, D-galactose, L-arabitol, D-fucose, and dulcitol, whereas D-lyxose isomerase was induced by D-lyxose, D-mannose, D-ribose, dulcitol, and myoinositol. Some compounds such as dulcitol, D-galactose, and D- or L-fucose which do not support growth are still able to serve as inducers for various pentose isomerases.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule.     

Use of chemostat for selection ofStreptomyces chrysomallus mutants altered in the induction of D-glucose isomerase

Summary D-glucose isomerase ofStreptomyces chrysomallus PL45 is inducible by D-xylose only. In mutants obtained by means of a selection procedure in a chemostat the isomerase was induced in xylose-free medium containing glucose as carbon source.     

Fermentation of D-xylose by yeasts using glucose isomerase in the medium to convert D-xylose to D-xylulose

Summary A method to obtain the fermentative conversion by yeasts of D-xylose to ethanol is described. The method depends on a combination of two factors; (1) the ability of glucose isomerase to isomerise D-xylose to D-xylulose and (2) the ability of a number of yeasts to ferment D-xylulose.     

A novel xylose isomerase from Neurospora crassa

Summary Highest production of xylose Isomerase by Neurospora crassa grown with different carbon sources was at 0.014 U mg^-1 with D-xylose. The enzyme exhibited maximum activity at pH 8.0 and 70°C and retained 100% activity at 45°C for 30 min at pH 8.0. It was activated by 8 mM Mg²⁺ whereas 2 mM Co²⁺ afforded protection against inactivation by heat. The K _m for xylose was 10 mM and 22 mM for xylose Isomerase and xylose reductase respectively at 28°C and pH 7.0. This is the first report on the presence of xylose isomerase in N. crassa and the existence of two different pathways for the utilization of D-xylose.     

Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by <Emphasis Type="Italic">Lactobacillus bifermentans</Emphasis>

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, and incubation period) and culture medium composition (mineral salts, carbon source, and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. For optimal growth, the strain required Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g., ammonium citrate). The bacterium had no requirement for sodium acetate for either growth or production of isomerases. The production rate of enzymes was increased when metal ions were added, primarily manganese (2.5 mM). The text was submitted by the authors in English.