NMR Application Probes a Novel and Ubiquitous Family of Enzymes That Alter Monosaccharide Configuration

Abstract

By exploiting nuclear magnetic resonance (NMR) techniques along with novel applications of saturation difference analysis, we deciphered the functions of the previously uncharacterized products of three bacterial genes, rbsD, fucU, and yiiL, which are part of the ribose, fucose, and rhamnose operons of Escherichia coli, respectively. We show that RbsD catalyzes the pyran to furan conversion of ribose, whereas FucU and YiiL are involved in the catalysis of the anomeric conversion of their respective sugars. It was observed that the anomeric exchange of only ribofuranose, not ribopyranose, occurs spontaneously in solution rationalizing its evolutionary incorporation into the nucleic acid. The RbsD and FucU proteins share sequence homology and belong to the same protein family that is found from eubacteria to human, whereas the YiiL homologues exist in archaebacteria and lower eukaryotes. These enzymes, including the galactose mutarotase, exhibit a certain degree of cross-specificity to structurally analogous sugars thereby encompassing all existing monosaccharides in terms of their reactivities. The ubiquitous presence of enzymes involved in the anomeric changes of monosaccharides highlights an importance of these activities in various cellular processes requiring efficient monosaccharide utilization.The ␣-␤ anomeric change of a monosaccharide involving a pyran configuration is considered very slow, e.g. the rate of glucose mutarotation is 0.015 min Ϫ1 in water (1). The only known enzyme for such a process is galactose mutarotase (GalM of Escherichia coli), which accelerates the conversion between the ␣-and ␤-anomers of D-glucose and D-galactose (2-4) presumably by enhancing the efficiency of glycolysis. The enzymes involving sugar metabolism including glycosylation tend to be selective for the ␣-or ␤-anomer, and thus a slow anomeric conversion may cause a problem in utilizing sugar. There has been no report on proteins involving an ␣-␤ conversion of sugars other than glucose, perhaps because of the lack of a method for detecting mutarotation at equilibrium. The conventional methods can only detect a change in optical rotation caused by an equilibrium shift from either the ␣-or ␤-anomer, which requires a purified ␣-or ␤-anomer as a substrate (3).Monosaccharides in general have ␣-and ␤-anomers of pyranose (six-membered ring, e.g. L-fucose and L-rhamnose) and often have additional ␣-and ␤-anomers of furanose (five-membered ring). D-Ribose, a key sugar moiety for the component of nucleic acids and ATP, exists in four different forms in solution, ␣-pyranose (22%), ␤-pyranose (58%), ␣-furanose (7%), and ␤-furanose (12%), with its open-chain form constituting less than 1% of the total ribose in solution. Ribose is transported as a ␤-D-pyranoribose, which is the major form in solution and binds to the ribose-binding protein (RbsB) in the bacterial periplasm (5). However, the ribokinase (RbsK) involved in the next step of ribose metabolism recognizes ␣-D-ribofuranose as a substrate (6). An apparent discrepancy in the subs...

RhaU of Rhizobium leguminosarum Is a Rhamnose Mutarotase

“…Mutarotases facilitate the interconversion of ␣ and ␤ anomers where the stereochemically less-favored anomer is required for a subsequent step in a catabolic sequence. Three such examples have so far been identified, including L-rhamnose mutarotase (YiiL in E. coli) (30, 31), galactose mutarotase (GalM) (5,6,8,9,14,36,37,38,39), and fucose mutarotase (FucU) (16,30). In this report, we characterize rhaU from R. leguminosarum and provide evidence that is consistent with the hypothesis that RhaU is an L-rhamnose mutarotase.…”

“…Sequence similarity (41% identity) suggested that RhaU was related to a group of hypothetical proteins that included YiiL of E. coli (28), which was recently characterized as an L-rhamnose mutarotase, catalyzing the ␣-to ␤-anomeric conversion of L-rhamnose (30,31). Based on the phenotype produced by yiiL in E. coli, it was expected that a rhaU-containing mutant would grow normally at high concentrations of L-rhamnose (0.2%) and more slowly at low concentrations (0.03%), but Rlt243 exhibited a slow-growth phenotype even at high L-rhamnose concentrations ( Table 3).…”

Ribose Utilization with an Excess of Mutarotase Causes Cell Death Due to Accumulation of Methylglyoxal

“…In a previous study (13), it was shown that the RbsD protein was required for growth on ribose when the sugar was transported through a mutated glucose transporter (PtsG). Recently, RbsD was characterized as a novel type of mutarotase involved in the anomeric conversion of ribose (16). Here, we report a growth inhibition phenotype of RbsD when the rbsD and rbsACBK genes were overproduced on separate plasmids (Fig.…”

“…This is particularly evident in cells transporting ribose at a lower affinity (13). It was found that monosaccharide mutarotases, including RbsD, are ubiquitous and conserved in both prokaryotes and eukaryotes (16). Intracellular production of MG is achieved in E. coli and most bacteria by MG synthase encoded by the mgs gene (20).…”

Structure-based Functional Annotation

“…This was confirmed by the STD spectra recorded for these three sugars (data not shown). Second, enzyme catalyzed anomeric interconversion was investigated using two- (32). Exchange cross-peaks between ␣ and ␤ Glc6P anomers could only be observed in the presence of YMR099cp enzyme (Fig.…”