Entamoeba histolytica: molecular characterization of an aldose 1-epimerase (mutarotase)

In cells, the alpha-anomers of aldoses are the preferred metabolizable substrates, while beta-anomers of aldoses play their role in glycan structure. In the cytoplasm, alpha- and beta-anomers of aldoses interconvert through the enzyme termed aldose 1-epimerase or mutarotase (EC 5.1.3.3). We have identified a mutarotase gene in Entamoeba histolytica, the causative agent of non-bacterial dysentery in humans. Cloning and characterization of this gene in two strains of the parasite (HM-1:IMSS and Rahman) that differ in their pathogenicity, revealed that the sequence is identical in both strains. A recombinant E. histolytica mutarotase was produced as well as specific antibodies that recognized a 38 kDa protein in trophozoite lysates of both strains. Mutarotase activity was observed with the recombinant protein as well as in lysates of both HM-1:IMSS and Rahman, the former exhibiting a slightly higher mutarotase activity. Finally, we have shown by complementation that overexpression of the E. histolytica mutarotase in a mutarotase defective Escherichia coli strain restores the ability of these bacteria to grow in minimal medium with phenyl-beta-galactopyranoside as the sole carbon source.     

A biosensor for the determination of amylase activity

A new biosensing flow injection method for the determination of alpha-amylase activity has been introduced. The method is based on the analysis of maltose produced during the hydrolysis of starch in the presence of alpha-amylase. Maltose determination in the flow system was allowed by the application of peroxide electrode equipped with an enzyme membrane. The membrane was obtained by immobilisation of glucose oxidase, alpha-glucosidase and optionally mutarotase on a cellophane, co-crosslinked by gelatin-glutaraldehyde together with bovine serum albumine. alpha-Glucosidase hydrolyses maltose to alpha-D-glucose, which is converted to beta-D-glucose by mutarotase. beta-D-Glucose is then determined via glucose oxidase. The new biosensor has the limit of detection of 50 nmol l(-1) maltose, which means 2 nkat ml(-1) in alpha-amylase activity units, when the reaction time of amylase was 5 min (determined with respect to a signal-to-noise ratio 3:1). When the reaction time of alpha-amylase was 30 min, the limit of detection was 0.5 nkat ml(-1). A linear range of current response was 0.1-3 mmol l(-1) maltose, with a response time of 35s. The biosensor was stable at least two months and retained 70% of its original activity (with mutarotase the stability is decreased to 3 weeks). When the enzyme membrane was stored in a dry state at 4 degrees C in a refrigerator, the lifetime was approximately 6 months (with mutarotase only 3 months).     

Molecular characterization of a ribosome-associated Hsp70-homologous gene from Rhizopus nigricans

A ribosome-associated Hsp70-homologous gene (Rnssb-1) was isolated from the genomic library of the filamentous zygomycete fungus Rhizopus nigricans. The nucleotide sequence of a genomic clone encoded the N-terminal part of a protein with high similarity to the yeast SSB ribosome-associated chaperones. The missing 3' end of the gene was obtained by 3' RACE. The Northern blot analysis showed that the Rnssb-1 gene is constitutively expressed and is not induced upon heat shock at 37 degrees C. The primary structure analyses revealed that the coding region of the Rnssb-1 gene is interrupted by at least four introns. Their splicing was not inhibited by exposure of the organism to heat shock as proven by RT-PCR. A Southern blot analysis of R. nigricans genomic DNA confirmed the presence of two additional gene copies of ribosome-associated Hsp70 genes in the fungal genome.     

Characterization of the Saccharomyces cerevisiae galactose mutarotase/UDP-galactose 4-epimerase protein, Gal10p

Saccharomyces cerevisiae and some related yeasts are unusual in that two of the enzyme activities (galactose mutarotase and UDP-galactose 4-epimerase) required for the Leloir pathway of d-galactose catabolism are contained within a single protein-Gal10p. The recently solved structure of the protein shows that the two domains are separate and have similar folds to the separate enzymes from other species. The biochemical properties of Gal10p have been investigated using recombinant protein expressed in, and purified from, Escherichia coli. Protein-protein crosslinking confirmed that Gal10p is a dimer in solution and this state is unaffected by the presence of substrates. The steady-state kinetic parameters of the epimerase reaction are similar to those of the human enzyme, and are not affected by simultaneous activity at the mutarotase active site. The mutarotase active site has a strong preference for galactose over glucose, and is not affected by simultaneous epimerase activity. This absence of reciprocal kinetic effects between the active sites suggests that they act independently and do not influence or regulate each other.     

Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase.

The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-galM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels.     

A continuous spectrophotometric assay for the enzymatic marker glucose 6-phosphatase

A continuous spectrophotometric assay for glucose 6-phosphatase is described. The method uses glucose dehydrogenase and mutarotase as ancillary enzymes. Glucose 6-phosphatase activity is measured by following NADH formation at 340 nm. The method is linear, at least up to 38 mU in the test which corresponds to a delta E of 0.24 min-1, when the enzyme is assayed in a microsomal fraction. We also discuss the method's suitability for subcellular fractionation. No other continuous assay for this important enzymatic marker of the endoplasmic reticulum is currently available.     

UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity.

UDPgalactose 4-epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N-terminal domain (residues 1-377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-epimerase, and a C-terminal domain (residues 378-699), which shows extensive identity to either the bacterial or human aldose 1-epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified epimerase preparations showed mutarotase activity and could convert either alpha-d-glucose or alpha-d-galactose to their beta-anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5'-UMP and l-arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-epimerase is a bifunctional enzyme with aldose 1-epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.     

Anomer specificity of glucose-6-phosphatase and glucokinase

The anomeric form of glucose produced by glucose-6-phosphatase was studied using an apparatus that specifically measures beta-D-glucose. The time course of beta-D-glucose formation from glucose-6-P by glucose-6-phosphatase is essentially linear. In the presence of mutarotase, this rate is reduced to 70% of that obtained in the absence of mutarotase. When detergent treated microsomes were used, the rate of beta-D-glucose formation is unaffected by mutarotase. These results suggest that only beta-anomer of glucose is produced by microsomal glucose-6-phosphatase and this specificity is determined by translocase for glucose-6-P or glucose. It was also demonstrated that alpha-D-glucose is the substrate for glucokinase.     

Enzymic hydrolysis of the carbon–fluorine bond of α-d-glucosyl fluoride by rat intestinal mucosa: Localization of intestinal maltase

1. alpha-d-Glucosyl fluoride was hydrolysed by an extract of rat intestinal mucosa. The pH optimum was 6.6 and the K(m) 0.4mm at 20 degrees . Activity was assayed by release of either glucose or fluoride. 2. The alpha-d-glucosyl fluoride-hydrolase activity of the extract was associated with both mutarotase and alpha-d-glucosidase activities. 3. Tris (5mm) inhibited both the alpha-d-glucosidase and alpha-d-glucosyl fluoride-hydrolase activities by 55% but did not inhibit mutarotase. The K(i) of tris for both enzyme activities was 2mm. 4. The extract did not hydrolyse melibiose and lactose. Mutarotase used both alpha-d-glucose and beta-l-arabinose as substrates but the glucosyl fluoride-hydrolase activity did not extend to beta-l-arabinosyl fluoride. 5. The thermal stability of alpha-d-glucosidase and alpha-d-glucosyl fluoride hydrolase was identical. Mutarotase was more thermolabile. 6. A preparation of the brush border of intestinal epithelial cells contained both alpha-d-glucosyl fluoride-hydrolase and alpha-d-glucosidase activities. In each precipitate and washing the ratio of the two activities was the same. All the mutarotase activity was in the first supernatant. 7. Agidex, a fungal amyloglucosidase, cleaved glucosyl fluoride in addition to maltose. Tris inhibited both activities and in each case the K(i) was 3mm. 8. The probable identity of alpha-d-glucosyl fluoride hydrolase with alpha-d-glucosidase is discussed and a possible mechanism for the reaction suggested. 9. Incubation of intestinal slices with alpha-d-glucosyl fluoride led to complete hydrolysis in 30min. The glucose rapidly entered the cell and was metabolized, leaving the fluoride in the incubation medium. This constitutes a further proof that the intestinal alpha-d-glucosidase, although on the brush border, is located outside the site of active transport of sugars.     

Genetic evidence for a defective xylan degradation pathway in Lactococcus lactis

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974-3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a beta-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.     

Acinetobacter calcoaceticus encoded mutarotase: nucleotide sequence analysis of the gene and characterization of its secretion in Escherichia coli.

The nucleotide sequence of the mutarotase gene from Acinetobacter calcoaceticus has been determined. It reveals an open reading frame of 381 amino acids. The codon usage of A. calcoaceticus for this gene is similar to E. coli except for the amino acids Leu, Ala, Glu, and Arg where major differences exist. This did not interfere drastically with high level expression in E. coli. The regulatory sequences for the initiation of translation are similar to the ones described for E. coli. The N-terminal 20 amino acids, which are not found in the mature enzyme, show homology to signal sequences of exported proteins. In A. calcoaceticus and E. coli mutarotase is specifically secreted into the periplasmic space. Processing of the signal sequence occurs at identical sites in both organisms. The mature mutarotase consists of 361 amino acids and has a calculated molecular weight of 38457 Da. Expression of mutarotase at a high level in a recombinant E. coli destabilizes the outer membrane. This results in coordinated leakage of mutarotase and beta-lactamase into the culture broth.     

Mutarotase in galactose-induced baker's yeast

Cell-free extracts of baker's yeast possess mutarotase activity only after induction of cells in the presence of galactose. The mutarotase activity appears 1 h after transfer to a galactose-containing medium and rises in synchrony with the utilization of galactose. Cycloheximide blocks the induction completely at a concentration of 100 μg/ml. InSaccharomyces fragilis the mutarotase is constitutive but its activity is strikingly increased after growth on galactose. The yeast mutarotase resembles in some respects analogous enzymes from other cells (pH dependence, substrate specificity, heat lability). Its affinity ford-galactose is substantially greater than ford-glucose. There may exist a coupling between mutarotase activity and the anomer-specific galactokinase.     

Galactose transporters discriminate steric anomers at the cell surface in yeast

Aldose-1-epimerase or mutarotase (EC 5.1.3.3) catalyzes interconversion of α/β-anomers of aldoses, such as glucose and galactose, and is distributed in a wide variety of organisms from bacteria to humans. Nevertheless, the physiological role of this enzyme has been elusive in most cases, because the α-form of aldoses in the solid state spontaneously converts to the β-form in an aqueous solution until an equilibrium of α : β=36.5 : 63.5 is reached. A gene named GAL10 encodes this enzyme in yeast. Here, we show that the GAL10 -encoded mutarotase is necessary for utilization of galactose in the milk yeast Kluyveromyces lactis , and that this condition is presumably created by the presence of the β-specific galactose transporter, which excludes the α-anomer from the α/β-mixture in the medium at the cell surface. Thus, we found that a mutarotase-deficient mutant of K. lactis failed to grow on medium, in which galactose was the sole carbon source, but, surprisingly, that the growth failure is suppressed by concomitant expression of the Saccharomyces cerevisiae -derived galactose transporter Gal2p, but not by that of the K. lactis galactose transporter Hgt1p. We also suggest the existence of another mutarotase in K. lactis , whose physiological role remains unknown, however.     

A new continuous optical assay for maltase and sucrase.

A new method for the assay of maltase and sucrase is reported. The method makes use of mutarotase, hexokinase and glucose 6-phosphate dehydrogenase as ancillary enzymes. The reaction is linear at least up to a delta E/min of 0.13.     

Characterization and role of fucose mutarotase in mammalian cells

L-Fucose for mammalian glycosylation contains an anomeric carbon atom generating alpha- and beta-L-fucoses. Based on sequence comparison of mouse and human homologs with the prokaryotic fucose mutarotases (FucU) characterized previously, we investigated their function in mammalian cells. By nuclear magnetic resonance (NMR) measurement with saturation difference analysis, the purified mammalian mutarotases were demonstrated to be involved in an interconversion between the two anomeric forms with comparable efficiency as that of the Escherichia coli FucU. The mouse gene was widely expressed in various tissues and cell lines, including kidney, liver, and pancreas, although expression was marginal in muscle and testis. By generating stably expressed cell lines for mutarotase genes in HepG2, it was shown that fucose incorporations into cellular proteins were increased as demonstrated by an incorporation of radiolabeled fucose into the cells. Furthermore, intracellular levels of GDP-L-fucose, measured with high performance liquid chromatography (HPLC), were enhanced by an overproduction of cellular mutarotase, which was reversed by gene silencing of mutarotase based on RNA interference. The results suggest that the mammalian mutarotase is functional in facilitated incorporation of fucose through the salvage pathway.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis. Evidence for independent mutarotation site.

UDP-galactose 4-epimerases from the yeast Kluyvero-myces fragilis and Escherichia coli are both homodimers but the molecular mass of the former (75 kDa/subunit) is nearly double that of the latter (39 kDa/subunit). Protein databank sequence homology revealed the possibility of mutarotase activity in the excess mass of the yeast enzyme. This was confirmed by three independent assay protocols. With the help of specific inhibitors and chemical modification reagents, the catalytic sites of epimerase and mutarotase were shown to be distinct and independent. Partial proteolysis with trypsin in the presence of specific inhibitors, 5'-UMP for epimerase and galactose for mutarotase, protected the respective activities. Similar digestion with double inhibitors cleaved the molecule into two fragments of 45 and 30 kDa. After separation by size-exclusion HPLC, they manifested exclusively epimerase and mutarotase activities, respectively. Epimerases from Kluyveromyces lactis var lactis, Pachysolen tannophilus and Schizosaccharomyces pombi also showed associated mutarotase activity distinct from the constitutively formed mutarotase activity. Thus, the bifunctionality of homodimeric yeast epimerases of 65-75 kDa/subunit appears to be universal. In addition to the inducible bifunctional epimerase/mutarotase, K. fragilis contained a smaller constitutive monomeric mutarotase of approximately 35 kDa.     

The autosomal dominant trait of obesity, acanthosis nigricans, hypertension, ischemic heart disease and diabetes type 2.

OBJECTIVE: Among obese subjects, acanthosis nigricans in both males and females is not as uncommon as previously thought. Whereas this finding was extensively evaluated in females, mostly in the context of polycystic ovaries syndrome, little attention has been paid to obese males with acanthosis nigricans. As acanthosis seems to be a marker for insulin resistance, the present study was designed to evaluate the hypothesis that the clinical syndrome of obesity and acanthosis would take a different clinical course than that of simple obesity. METHODS: To characterize the course of acanthosis nigricans and obesity in males, we examined 22 children and adolescents with this complex, together with their parents and grandparents and found them to follow a detrimental sequence of the metabolic syndrome. We compared the findings to 13 age-matched males with obesity but no clinical apparent acanthosis nigricans. We analyzed the clinical course, fat distribution, glucose, insulin and C-peptide and lipoproteins. RESULTS: Onset of obesity in the metabolic syndrome group was at a mean age of 6.4 years, as compared to 2.3 years in the controls. The metabolic syndrome patients had a truncal (android) distribution of fat and their fasting blood glucose was significantly higher. HDL/total cholesterol was lower. Examination of the pedigrees suggested autosomal dominant inheritance of the obesity and acanthosis nigricans complex, extending to hypertension and ischemic heart disease in the parents' generation, and further extending to include diabetes type 2 in the grandparents' generation. CONCLUSIONS: This metabolic syndrome is inherited as an autosomal dominant trait, with onset of truncal obesity at age 6-7 years, acanthosis nigricans during childhood or adolescence, extending to hypertension and ischemic heart disease during young adulthood, and further extending to include diabetes type 2 in late adulthood. It is recommended that such children should be followed up as an 'at-risk' group, and would probably benefit from intensive weight reduction, which may prevent the later manifestations of the syndrome.