Dihydroxyacetone (Dha) kinases are a family of sequence-conserved enzymes which utilize either ATP (in animals, plants and eubacteria) or phosphoenolpyruvate (PEP, in eubacteria) as their source of high-energy phosphate. The kinases consist of two domains/subunits: DhaK, which binds Dha covalently in hemiaminal linkage to the Nepsilon2 of a histidine, and DhaL, an eight-helix barrel that contains the nucleotide-binding site. The PEP-dependent kinases comprise a third subunit, DhaM, which rephosphorylates in situ the firmly bound ADP cofactor. DhaM serves as the shuttle for the transfer of phosphate from the bacterial PEP: carbohydrate phosphotransferase system (PTS) to the Dha kinase. The DhaL and DhaK subunits of the PEP-dependent Escherichia coli kinase act as coactivator and corepressor of DhaR, a transcription factor from the AAA(+) family of enhancerbinding proteins. In Gram-positive bacteria genes for homologs of DhaK and DhaL occur in operons for putative transcription factors of the TetR and DeoR families. Proteins with the Dha kinase fold can be classified into three families according to phylogeny and function: Dha kinases, DhaK and DhaL homologs (paralogs) associated with putative transcription regulators of the TetR and DeoR families, and proteins with a circularly permuted domain order that belong to the DegV family.

Bacterial dihydroxyacetone (Dha) kinases do not exchange the ADP for ATP but utilize a subunit of the phosphoenolpyruvate carbohydrate phosphotransferase system for in situ rephosphorylation of a permanently bound ADP-cofactor. Here we report the 2.1-Å crystal structure of the transient complex between the phosphotransferase subunit DhaM of the phosphotransferase system and the nucleotide binding subunit DhaL of the Dha kinase of Lactococcus lactis, the 1.1-Å structure of the free DhaM dimer, and the 2.5-Å structure of the Dha-binding DhaK subunit. Conserved salt bridges and an edge-to-plane stacking contact between two tyrosines serve to orient DhaL relative to the DhaM dimer. The distance between the imidazole N⑀2 of the DhaM His-10 and the ␤-phosphate oxygen of ADP, between which the ␥-phosphate is transferred, is 4.9 Å . An invariant arginine, which is essential for activity, is appropriately positioned to stabilize the ␥-phosphate in the transition state. The (␤␣) 4 ␣ fold of DhaM occurs a second time as a subfold in the DhaK subunit. By docking DhaL-ADP to this subfold, the nucleotide bound to DhaL and the C1-hydroxyl of Dha bound to DhaK are positioned for in-line transfer of phosphate.Dihydroxyacetone (Dha) 4 is the structurally simplest among all metabolized carbohydrates. It is formed as an intermediate of methanol assimilation by methylotrophic yeast (1) and as the end product of dissimilative glycerol oxidation in the respiratory chain of Gluconobacter (2). Dha is taken up and metabolized by bacteria and eukaryotes. The first step of Dha metabolism is either a reduction to glycerol followed by glycerol phosphorylation or, alternatively, direct phosphorylation to the glycolytic intermediate Dha-P. There are two kinases that phosphorylate Dha, glycerol kinase and Dha kinase. Glycerol kinase does not discriminate between glycerol and Dha. In contrast, Dha kinases are specific for Dha, D-glyceraldehyde, and possibly other short-chain aldehydes and ketones. The chemical basis for this specificity is the formation of a covalent hemiaminal bond between the carbonyl carbon of Dha and the N⑀2 of the active-site histidine (for a review see Ref.3).Dha kinases can be grouped in two structurally homologous but functionally different families. The first are the two-domain Dha kinases (DAK) occurring in eukaryotes and some bacteria. They consist of a Dha binding (K) and an ATP binding (L) domain (4). Characteristically for kinases, ADP is exchanged by ATP after each catalytic cycle. ATP O ¡ DAK Dha REACTION 1The second family comprises Dha kinases that occur only in eubacteria and a few archaebacteria. These kinases consist of three subunits (5). Two of them, DhaK and DhaL, are homologous to the K and L domains. Intriguingly, the ADP moiety is not exchanged for ATP (6) but remains permanently bound to the DhaL subunit where it is rephosphorylated in situ by the third subunit, DhaM. These Dha kinases function according to Reaction 2.Phosphate is transferred sequentially from PEP via Enzyme I to HPr, hence to DhaM,...

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...

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