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

Abstract

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 ATPdependent 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-speci®c transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.

Aerobic glycerol dissimilation via the Enterococcus faecalis DhaK pathway depends on NADH oxidase and a phosphotransfer reaction from PEP to DhaK via EIIADha

“…BLAST analysis using the protein sequence of the small hypothetical polypeptide (EF1359) of the dhaK operon against the genome sequences of E. coli MG1655 and L. lactis IL1403 revealed around 30 % sequence identity with the N-terminal part (residues 1-128) of the multidomain DhaM EIIA-HPr-EI E. coli protein and DhaM Ll of L. lactis. The N terminus of the E. coli protein is similar to the Nterminal domain of the EIIAB Man subunit of the E. coli mannose transporter and contains a phosphorylatable histidine (Gutknecht et al, 2001), which is also conserved in DhaM Ll and EF1359. We therefore speculated that the …”

“…P~EIIB transfers its phosphoryl group to the carbohydrate bound to the corresponding membrane-integrated EIIC, and the resulting P-sugar is subsequently released into the cytoplasm (Deutscher et al, 2006). In addition, an involvement of the PTS in the transport-independent phosphorylation of dihydroxyacetone (DHA) generated during glycerol catabolism has been demonstrated in Escherichia coli (Gutknecht et al, 2001). Glycerol can be dissimilated by two biochemical modes in bacteria (Lin, 1976).…”

“…The PEP-dependent enzymes are specific to eubacteria. The phosphotransfer cascade has been well studied in E. coli (Gutknecht et al, 2001). The dhaK operon in this species comprises the three genes dhaK, dhaL and dhaM.…”

“…The dhaK operon in this species comprises the three genes dhaK, dhaL and dhaM. The first two genes encode the two subunits of DHA kinase and the third a multidomain polypeptide with an EIIA Man -like domain followed by an HPr-like domain and a domain showing homology to the N-terminal part of EI (Gutknecht et al, 2001). Based on genetic and in vitro phosphorylation experiments it has been concluded that the phosphoryl group of PEP is sequentially transferred via EI and HPr to the EI domain of DhaM.…”

“…Due to a DhaM intramolecular phosphorelay, the phosphate group is then transferred to the HPr-like and subsequently to the EIIA Man -like domain. The final transfer steps involve phosphotransfer from DhaM to the ADP cofactor of the DhaL subunit of DHA kinase and from the presumed DhaL/ATP intermediate to DHA bound to the DhaK subunit (Gutknecht et al, 2001) (see Fig. 1).…”