Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize either ATP (in animals, plants, bacteria) or the bacterial phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) as a source of high-energy phosphate. The PTS-dependent kinase of Escherichia coli consists of three subunits: DhaK contains the Dha binding site, DhaL contains ADP as cofactor for the double displacement of phosphate from DhaM to Dha, and DhaM provides a phospho-histidine relay between the PTS and DhaL::ADP. DhaR is a transcription activator belonging to the AAA+ family of enhancer binding proteins. It stimulates transcription of the dhaKLM operon from a sigma70 promoter and autorepresses dhaR transcription. Genetic and biochemical studies indicate that the enzyme subunits DhaL and DhaK act antagonistically as coactivator and corepressor of the transcription activator by mutually exclusive binding to the sensing domain of DhaR. In the presence of Dha, DhaL is dephosphorylated and DhaL::ADP displaces DhaK and stimulates DhaR activity. In the absence of Dha, DhaL::ADP is converted by the PTS to DhaL::ATP, which does not bind to DhaR.

Thermoanaerobacter tengcongensis is a thermophilic eubacterium that has a phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) of 22 proteins. The general PTS proteins, enzyme I and HPr, and the transporters for N-acetylglucosamine (EIICB(GlcNAc)) and fructose (EIIBC(Fru)) have thermal unfolding transitions at ∼90 °C and a temperature optimum for in vitro sugar phosphotransferase activity of 65 °C. The phosphocysteine of a EIICB(GlcNAc) mutant is unusually stable at room temperature with a t(1/2) of 60 h. The PEP binding C-terminal domain of enzyme I (EIC) forms a metastable covalent adduct with PEP at 65 °C. Crystallization of this adduct afforded the 1.68 Å resolution structure of EIC with a molecule of pyruvate in the active site. We also report the 1.83 Å crystal structure of the EIC-PEP complex. The comparison of the two structures with the apo form and with full-length EI shows differences between the active site side chain conformations of the PEP and pyruvate states but not between the pyruvate and apo states. In the presence of PEP, Arg465 forms a salt bridge with the phosphate moiety while Glu504 forms salt bridges with Arg186 and Arg195 of the N-terminal domain of enzyme I (EIN), which stabilizes a conformation appropriate for the in-line transfer of the phosphoryl moiety from PEP to His191. After transfer, Arg465 swings 4.8 Å away to form an alternative salt bridge with the carboxylate of Glu504. Glu504 loses the grip of Arg186 and Arg195, and the EIN domain can swing away to hand on the phosphoryl group to the phosphoryl carrier protein HPr.

Maturation of human intestinal lactase-phlorizin hydrolase (LPH) requires that a precursor (pro-LPH) be proteolytically processed to the mature microvillus membrane enzyme (m-LPH). Tlxe subeellular site of this processing is unknown. Using low.temperature experiments and brefeldin A (BFA), intmcellular transport was blocked in intestinal epithelial cells. In Caco-2 cells incubated at 180C, pro-LPH was complex-glycosylated but not cleaved, while at 20°C small amounts of proteolytically processed LPH were observed. These data exclude a pre-Golgi proteolytic event. BFA completely blocked proteolytie maturation of LP H and lead to an aberrant form of pro-LPH in both Caco*2 cells and intestinal explants. Therefore, proteolytic processing of LPH is a postoGolgi event, oceuring either in the trans.Golgi network, transport vesicles, or after insertion of pro-LPH into the microvillus membrane,

Dihydroxyacetone kinases are a family of sequencerelated enzymes that utilize either ATP or a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as a source of high energy phosphate. The PTS is a multicomponent system involved in carbohydrate uptake and control of carbon metabolism in bacteria. Phylogenetic analysis suggests that the PTS-dependent dihydroxyacetone kinases evolved from an ATPdependent ancestor. Their nucleotide binding subunit, an eight-helix barrel of regular up-down topology, retains ADP as phosphorylation site for the double displacement of phosphate from a phospho-histidine of the PTS protein to dihydroxyacetone. ADP is bound essentially irreversibly with a t1 ⁄2 of 100 min. Complexation with ADP increases the thermal unfolding temperature of dihydroxyacetone L from 40 (apo-form) to 65°C (holoenzyme). ADP assumes the same role as histidines, cysteines, and aspartic acids in histidine kinases and PTS proteins. This conversion of a substrate binding site into a cofactor binding site reflects a remarkable instance of parsimonious evolution.Few compounds are as ubiquitous and highly connected as adenine nucleotides (1). ATP functions as a carrier of chemical energy, ADP, AMP, ADP-ribosyl, adenylyl moieties as enzyme regulators, and cAMP as a second messenger. The nucleotide coenzymes NADH, FAD, and coenzyme A contain an adenosyl group that without direct participation in catalysis assists in binding to the apoenzyme. Here we report on ADP acting as phosphorylation site in the double displacement phosphoryl transfer reaction catalyzed by the Escherichia coli dihydroxyacetone (Dha) 1 kinase. Dha kinases are a family of sequencerelated enzymes that can be divided into two groups according to their phosphate donor, namely ATP or a phosphoprotein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) (2) (Fig. 1A). ATP-dependent kinases occur in bacteria, yeast, animals, and plants; Dha kinases dependent on the PTS, a dedicated energy transducing system involved in carbohydrate uptake and control of carbon metabolism (3, 4), occur only in bacteria. In methylotrophic yeast, free Dha is the product of a transketolase reaction between ribulose-5-phosphate and formaldehyde derived from methanol. In bacteria, Dha is formed by oxidation of glycerol (5-7). For yeast it has been shown that Dha kinases fulfill a "housecleaning" function by removing chemically reactive (8) and potentially hazardous short chain carbohydrates (9).The Dha kinases of C. freundii (DAK) and of E. coli (DhaK, DhaL) are prototypes of ATP-and PTS-dependent kinase, respectively (2, 10, 11). The former consists of two domains that are connected by a long flexible linker, the latter of two subunits (DhaK and DhaL) that show homology with DAK throughout their combined lengths. DhaK contains the Dha binding site, DhaL the nucleotide binding site (12, 13). The DhaL fold, an eight-helix barrel of regular up-down topology, constitutes a new scaffold of nucleotide-binding proteins (Fig.

Dihydroxyacetone (Dha) kinases are a novel family of kinases with signaling and metabolic functions. Here we report the x-ray structures of the transcriptional activator DhaS and the coactivator DhaQ and characterize their function. DhaQ is a paralog of the Dha binding Dha kinase subunit; DhaS belongs to the family of TetR repressors although, unlike all known members of this family, it is a transcriptional activator. DhaQ and DhaS form a stable complex that in the presence of Dha activates transcription of the Lactococcus lactis dha operon. Dha covalently binds to DhaQ through a hemiaminal bond with a histidine and thereby induces a conformational change, which is propagated to the surface via a cantileverlike structure. DhaS binding protects an inverted repeat whose sequence is GGACACATN 6 ATTTGTCC and renders two GC base pairs of the operator DNA hypersensitive to DNase I cleavage. The proximal half-site of the inverted repeat partially overlaps with the predicted ؊35 consensus sequence of the dha promoter.The enzymes of metabolic pathways have by and large been conserved in all kingdoms of life where they occur. In contrast, the mechanisms of pathway control are diverse. This is most obvious at the level of gene expression. The different size, structure, and sequence organization of eukaryotic and prokaryotic genomes necessitate the different control mechanisms. But even between bacteria with similar genome organizations the differences can be striking. The transcription control of dihydroxyacetone kinases is one example for such diversity, as it will be shown below. Dihydroxyacetone (Dha)4 kinases occur in eubacteria, animals, and plants. They can be divided into two families according to the source of high energy phosphate they utilize, ATP and phosphoenolpyruvate (PEP) (for a review see Ref. 1). The ATPdependent kinases from animals, plants, and eubacteria consist of a Dha binding and an ATP binding domain. The PEP-dependent forms consist of three protein subunits DhaK, DhaL, and DhaM (2). DhaK and DhaL are homologous to the Dha and ATP binding domains and DhaM is homologous to the IIA Mansubunits of the PEP: sugar phosphotransferase system (PTS) (3, 4). DhaK is a stable homodimer of 35-kDa subunit molecular mass that binds Dha covalently by a hemiaminal linkage between the imidazole nitrogen of a histidine (His-230 in Escherichia coli) and the carbonyl carbon of Dha (5, 6). DhaL contains a molecule of ADP, which in contrast to the nucleotide of the ATP-dependent kinases is not exchanged but is rephosphorylated in situ by DhaM (7). DhaM shuttles phosphate from the phosphorylcarrier protein HPr of the PTS to DhaL (2).A BLAST analysis with DhaK and DhaL as query revealed genes for DhaK and DhaL homologs, which were associated in operons with the genes for putative transcription factors (1). These genes occur adjacent to the Dha kinase operons suggesting that they control Dha kinase expression. How this works has so far been elucidated only for E. coli (8). Here, the dha operon is controlled by DhaR, a trans...