Bernhard Erni

The dihydroxyacetone kinase of Escherichia coli utilizes a phosphoprotein instead of ATP as phosphoryl donor

The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N‐ and C‐terminal halves of the ATP‐dependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N‐terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C‐terminus is similar to the N‐terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI‐ and HPr‐dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate‐specific transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.

Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR

LevR, which controls the expression of the lev operon of Bacillus subtilis, is a regulatory protein containing an N‐terminal domain similar to the NifA/NtrC transcriptional activator family and a C‐terminal domain similar to the regulatory part of bacterial anti‐terminators, such as BglG and LicT. Here, we demonstrate that the activity of LevR is regulated by two phosphoenolpyruvate (PEP)‐dependent phosphorylation reactions catalysed by the phosphotransferase system (PTS), a transport system for sugars, polyols and other sugar derivatives. The two general components of the PTS, enzyme I and HPr, and the two soluble, sugar‐specific proteins of the lev‐PTS, LevD and LevE, form a signal transduction chain allowing the PEP‐dependent phosphorylation of LevR, presumably at His‐869. This phosphorylation seems to inhibit LevR activity and probably regulates the induction of the lev operon. Mutants in which His‐869 of LevR has been replaced with a non‐phosphorylatable alanine residue exhibited constitutive expression from the lev promoter, as do levD or levE mutants. In contrast, PEP‐dependent phosphorylation of LevR in the presence of only the general components of the PTS, enzyme I and HPr, regulates LevR activity positively. This phosphorylation most probably occurs at His‐585. Mutants in which His‐585 has been replaced with an alanine had lost stimulation of LevR activity and PEP‐dependent phosphorylation by enzyme I and HPr. This second phosphorylation of LevR at His‐585 is presumed to play a role in carbon catabolite repression.

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS).

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICBGlc subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICBGlc are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICBGlc and IIAGlc were produced in vivo by RecA intein‐mediated trans‐splicing. Purified, cyclized IIAGlc and IICBGlc had 100% and 30% of wild‐type glucose phosphotransferase activity, respectively. Cyclized IIAGlc displayed increased stability against temperature and GuHCl‐induced unfolding.

Escherichia coli dihydroxyacetone kinase controls gene expression by binding to transcription factor DhaR

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.

Small substrate, big surprise: fold, function and phylogeny of dihydroxyacetone kinases

Abstract.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 Nε2 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.

Crystal structure of the Citrobacter freundii dihydroxyacetone kinase reveals an eight-stranded alpha-helical barrel ATP-binding domain.

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 α/β-folds with a molecule of dihydroxyacetone covalently bound in hemiaminal linkage to the Nϵ2 of His-220. The C-terminal domain consists of a regular eight-helix α-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 Mg2+ ions to γ-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.

The glucose transporter of the Escherichia coli phosphotransferase system. Mutant analysis of the invariant arginines, histidines, and domain linker.

The glucose transporter of the bacterial phosphotransferase system (PTS) consists of a hydrophilic (IIAGlc) and a transmembrane subunit (IICBGlc). IICBGlc has two domains (C and B), which are linked by a highly invariant sequence. Transport of glucose by IIC and phosphorylation by IIB are tightly coupled processes. Three motifs that are strongly conserved in 12 homologous PTS transporters, namely two invariant arginines (Arg-424 and Arg-426) adjacent to the phosphorylation site (Cys-421), the invariant interdomain sequence KTPGRED, and two conserved histidines (His-211 and His-212) in the IIC domain were mutated and the mutant proteins characterized in vivo and in vitro for transport and phosphorylation activity. Replacement of the strongly β-turn favoring residues Thr and Gly of the linker by α-helix favoring Ala results in strong reduction of activity, whereas the substitutions of the other residues have only minor effects. The R424K and R426K mutants can be phosphorylated by IIAGlc but can no longer donate the phosphoryl group to glucose. The H211Q and H212Q mutants continue to phosphorylate glucose at a reduced rate but H212Q can no longer transport glucose. Mixtures of purified R424K/H212Q and R426K/H212Q have 10% of wild-type phosphorylation activity and when coexpressed inEscherichia coli support glucose transport.

A mechanism of covalent substrate binding in the x-ray structure of subunit K of the Escherichia coli dihydroxyacetone kinase

Dihydroxyacetone (Dha) kinases are homologous proteins that use different phosphoryl donors, a multiphosphoryl protein of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system in bacteria, ATP in animals, plants, and some bacteria. The Dha kinase of Escherichia coli consists of three subunits, DhaK and DhaL, which are colinear to the ATP-dependent Dha kinases of eukaryotes, and the multiphosphoryl protein DhaM. Here we show the crystal structure of the DhaK subunit in complex with Dha at 1.75 Å resolution. DhaK is a homodimer with a fold consisting of two six-stranded mixed β-sheets surrounded by nine α-helices and a β-ribbon covering the exposed edge strand of one sheet. The core of the N-terminal domain has an α/β fold common to subunits of carbohydrate transporters and transcription regulators of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system. The core of the C-terminal domain has a fold similar to the C-terminal domain of the cell-division protein FtsZ. A molecule of Dha is covalently bound in hemiaminal linkage to the Nε2 of His-230. The hemiaminal does not participate in covalent catalysis but is the chemical basis for discrimination between short-chain carbonyl compounds and polyols. Paralogs of Dha kinases occur in association with transcription regulators of the TetR/QacR and the SorC families, pointing to their biological role as sensors in signaling.

Regulation of the Dha Operon of Lactococcus lactis A DEVIATION FROM THE RULE FOLLOWED BY THE TetR FAMILY OF TRANSCRIPTION REGULATORS

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 cantilever-like structure. DhaS binding protects an inverted repeat whose sequence is GGACACATN6ATTTGTCC 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.

PHAGE DISPLAY SELECTION OF PEPTIDES AGAINST ENZYME I OF THE PHOSPHOENOLPYRUVATE-SUGAR PHOSPHOTRANSFERASE SYSTEM (PTS)

The bacterial phosphoenolpyruvate–sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates and is involved in signal transduction. In response to the availability of carbohydrates it modulates catabolite repression, intermediate metabolism, gene expression and chemotaxis. It is ubiquitous in bacteria but does not occur in animals and plants. Uniqueness and pleiotropic function make the PTS a target for new antibacterial drugs. Enzyme I is the first component of the divergent protein phosphorylation cascade of the PTS. It transfers phosphoryl groups from phosphoenolpyruvate to the general phosphoryl carrier protein HPr. Six 15‐mer, nine 10‐mer and nine 6‐mer peptides that inhibit enzyme I were selected from phage display libraries. Of these, 16 were synthesized and characterized. The majority of the peptides contain a histidine with an adjacent arginine. Two peptides were found to contain cysteines but no histidine. All peptides are rich in basic residues and lack acidic amino acids. The peptides inhibit the phosphotransferase system in vitro with IC50 of between 10 μM and 2 mM. Some, but not all, of the peptides inhibit cell growth in the agar diffusion test by an as yet undefined mechanism. All peptides are phosphorylated by enzyme I, and some are regenerated by slow autocatalytic hydrolysis of the phospho–peptide bond.