Deciphering the function of an ORF: Salmonella enterica DeoM protein is a new mutarotase specific for deoxyribose

We identified in Salmonella enterica serovar Typhi a cluster of four genes encoding a deoxyribokinase (DeoK), a putative permease (DeoP), a repressor (DeoQ), and an open reading frame encoding a 337 amino acid residues protein of unknown function. We show that the latter protein, called DeoM, is a hexamer whose synthesis is increased by a factor over 5 after induction with deoxyribose. The CD spectrum of the purified recombinant protein indicated a dominant contribution of betatype secondary structure and a small content of alpha-helix. Temperature and guanidinium hydrochloride induced denaturation of DeoM indicated that the hexamer dissociation and monomer unfolding are coupled processes. DeoM exhibits 12.5% and 15% sequence identity with galactose mutarotase from Lactococcus lactis and respectively Escherichia coli, which suggested that these three proteins share similar functions. Polarimetric experiments demonstrated that DeoM is a mutarotase with high specificity for deoxyribose. Site-directed mutagenesis of His183 in DeoM, corresponding to a catalytically active residue in GalM, yielded an almost inactive deoxyribose mutarotase. DeoM was crystallized and diffraction data collected for two crystal systems, confirmed its hexameric state. The possible role of the protein and of the entire gene cluster is discussed in connection with the energy metabolism of S. enterica under particular growth conditions.     

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.     

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.     

The catalytic mechanism of galactose mutarotase

Galactose mutarotase catalyzes the first step in normal galactose metabolism by catalyzing the conversion of beta-D-galactose to alpha-D-galactose. The structure of the enzyme from Lactococcus lactis was recently solved in this laboratory and shown to be topologically similar to domain 5 of beta-galactosidase. From this initial X-ray analysis, four amino acid residues were demonstrated to be intimately involved in sugar binding to the protein: His 96, His 170, Asp 243, and Glu 304. Here we present a combined X-ray crystallographic and kinetic analysis designed to examine the role of these residues in the reaction mechanism of the enzyme. For this investigation, the following site-directed mutant proteins were prepared: H96N, H170N, D243N, D243A, E304Q, and E304A. All of the structures of these proteins, complexed with either glucose or galactose, were solved to a nominal resolution of 1.95 A or better, and their kinetic parameters were measured against D-galactose, D-glucose, L-arabinose, or D-xylose. From these studies, it can be concluded that Glu 304 and His 170 are critical for catalysis and that His 96 and Asp 243 are important for proper substrate positioning within the active site. Specifically, Glu 304 serves as the active site base to initiate the reaction by removing the proton from the C-1 hydroxyl group of the sugar substrate and His 170 functions as the active site acid to protonate the C-5 ring oxygen.     

Characterization of the proteins encoded by the Bacillus subtilis yoxA-dacC operon

In Bacillus subtilis , the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli . The peptide d -Glu-δ- m -A₂pm- d -Ala- m -A₂pm- d -Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd -endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed.