Dihydroxyacetone kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system in bacteria. Here we report the 2.5-Å crystal structure of the homodimeric Citrobacter freundii dihydroxyacetone kinase complex with an ATP analogue and dihydroxyacetone. The N-terminal domain consists of two ␣/␤-folds with a molecule of dihydroxyacetone covalently bound in hemiaminal linkage to the N⑀2 of His-220. The Cterminal domain consists of a regular eight-helix ␣-barrel. The eight helices form a deep pocket, which includes a tightly bound phospholipid. Only the lipid headgroup protrudes from the surface. The nucleotide is bound on the top of the barrel across from the entrance to the lipid pocket. The phosphate groups are coordinated by two Mg 2؉ ions to ␥-carboxyl groups of aspartyl residues. The ATP binding site does not contain positively charged or aromatic groups. Paralogues of dihydroxyacetone kinase also occur in association with transcription regulators and proteins of unknown function pointing to biological roles beyond triose metabolism. Dihydroxyacetone (Dha)1 kinases can utilize either of two sources for high energy phosphate, ATP or a phosphoprotein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) (1). Little is known about the function of this enzyme and the metabolic origin of its substrate, Dha. Dihydroxyacetone phosphate (DhaP) can be formed by aldol cleavage of fructose-1,6-bisphosphate, by isomerization from glyceraldehyde-3-phosphate, and by oxidation of glycerol-3-phosphate in the mitochondrial glycerol phosphate shuttle. DhaP is an obligatory precursor of glyceryl ether phospholipid biosynthesis (2). Free Dha plays a pivotal role in methanol assimilation by methylotrophic yeast and plants where it is produced in the transketolase reaction between xylulose-5-phosphate and formaldehyde catalyzed by dihydroxyacetone synthase (3-6). Bacteria produce free Dha by oxidation of glycerol under anaerobic conditions and aldol cleavage of fructose-6-phosphate (9 -13). Dha is a carbon source for bacteria, and if added to the medium, it is also used as gluconeogenic precursor by mammalian tissues (14 -18). Although the pathways utilizing free Dha appear few and limited in scope, genes for Dha kinases and Dha kinase homologues are widely distributed among plants and animals where their biological function is not obvious (for a survey see accession numbers PF02733 and PF02734 at www.sanger.ac.uk/Software/Pfam/index.shtml). Dha like other triose sugars has an increased propensity to react with proteins in Maillard-type reactions (19,20), because unlike hexoses and pentoses, it cannot be deactivated by formation of cyclic hemiacetals. The chemical reactivity of Dha might be the rationale for its use as a therapeutic tanning agent (21, 22). It has recently been shown that Dha can be toxic to yeast cells and that detoxification is d...

Strains of For almost 50 years, methyl-␣-D-glucopyranoside (␣-MGlc)3 has served as a model substrate for probing the mechanism(s) of sugar transport in bacteria. Fortuitously (but importantly), ␣-MGlc is not metabolized by Escherichia coli K12, and transport data were interpretable without the ambiguities caused by further catabolism of the accumulated sugar. Early studies of ␣-MGlc uptake by cells of E. coli introduced the "permease" concept to bacterial physiologists, and "active transport" was incorporated in the lexicon of microbial energetics (1-5). The toxicity of ␣-MGlc toward E. coli was soon recognized, and Rogers and Yu (2) unexpectedly found that the glucose analog was recoverable from cells in both free and phosphorylated form. An understanding of these observations awaited the serendipitous discovery, and subsequent biochemical dissection, of the bacterial phosphoenolpyruvate-dependent sugar:phosphotransferase system (PEP-PTS; EC 2.7.1.69) by Roseman and co-workers (6, 7). The multicomponent PEP-PTS includes membrane-localized, sugar-specific transporters (enzymes IICB) that are fused or associated with a third domain (IIA) and two general cytoplasmic proteins (enzyme I and HPr). Collectively, these interactive components constitute a phospho-relay that (in five sequential stages) transfers the high energy phosphoryl moiety from PEP to catalyze the simultaneous phosphorylation and translocation of sugars through the cytoplasmic membrane (for reviews of PTS functions and nomenclature see . Completion of the chromosomal DNA sequence of E. coli K12 strain MG-1655 in 1997 (15), confirmed that ptsG encodes one (IICB Glc /PtsG) of two glucose-specific PTS transporters, whereas the genes for the general proteins and IIA Glc reside within a separate ptsHIcrr operon. After the discovery of other PEP-PT systems, it became apparent that ␣-MGlc was a reasonably specific substrate for the PtsG transporter in E. coli. , it was established that ␣-MGlc6P was accumulated via PtsG to toxic concentrations and that intracellular ␣-MGlc was formed via dephosphorylation of ␣-MGlc6P (Fig. 1, left).Our interest in the transport and bactericidal effects of ␣-MGlc(6P) in E. coli K12 stems from the fact that Klebsiella pneumoniae ATCC 23357 (a taxonomically close relative) readily ferments ␣-MGlc as an energy source for growth. Furthermore, and again in contrast with E. coli K12 strains, K. pneumoniae also metabolizes the following five O-␣-linked isomers of sucrose: trehalulose, turanose, maltulose, leucrose, and palatinose (20, 21). We presented evidence (21) for a tricistronic ␣-glucoside (Agl) operon comprising genes aglR, aglA, and aglB. We suggested that the three genes encode (in order), a transcriptional regulatory protein (AglR), an ␣-glucoside PTS transporter AglA (IICB Agl Swiss-Prot accession number Q9AGA7), and an Mn

Summary. Plasma samples of 1126 dogs belonging to 21 different European breeds were analysed by two‐dimensional agarose gel (pH 5.4 or pH 8.6) – horizontal polyacrylamide gel (pH 9.0) electrophoresis, followed by general‐protein staining of gels. Genetic polymorphism was detected for five as yet unidentified proteins designated pretransferrin‐1 and ‐2 (Prt1 and Prt2) and postalbumin‐1, ‐2 and ‐3 (Pa1, Pa2 and Pa3). Three alleles are reported in the Prt1 and Pa2 systems and two alleles in the Prt2, Pa1 and Pa3 systems. While Prt2 variation was observed only in the cocker spaniel breed, each of the other four proteins showed a high degree of polymorphism in most of the breeds studied. Pa3 fractions were clearly observed only in samples stored at ‐20°C for more than 2 years. Prt1, Pa1 and Pa2 proteins are additional useful markers for parentage control in dogs. This study corroborated previously published results that dog plasma proteins, in general, show considerably more polymorphism than that reported for haemoglobin and for several blood cell enzymes in this species.

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Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log 10 CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log 10 CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.