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

Abstract

In methylotrophic yeast, Dha is the primary product of methanol assimilation (1). In bacteria, Dha is formed by oxidation of glycerol or aldol cleavage of fructose-6-phosphate (2, 3). In animal cells, Dha is a gluconeogenic precursor (4-6). The Dha kinase of Escherichia coli was discovered in the proteome as two spots, which were up-regulated in the ptsI mutant, lacking enzyme I of the phosphoenolpyruvate(PEP)-dependent carbohydrate:phosphotransferase system (PTS) (7) and displayed strong amino acid sequence similarities to the N-and C-terminal domains of the ATP-dependent Dha kinase of Citrobacter freundii (7,8). The two subunits, termed DhaK and DhaL (SWISS-PROT entries P76015 and P76014), are encoded in an operon together with a third protein, DhaM (SWISS-PROT entry P37349). DhaM is a multiphosphoryl protein of the PTS with sequence similarity to the IIA domain of the mannose transporter (PDB ID code 1PDO; ref. No similarity with known protein folds could be predicted for DhaK and DhaL. DhaK, DhaL, and DhaM were overexpressed and purified (12). Whereas DhaL precipitates already at low protein concentration, DhaK is soluble. Here we describe the x-ray structure of DhaK with Dha covalently bound to a histidine in the active site. Materials and MethodsProtein Purification and Activity Assay. E. coli DhaK was overproduced in E. coli WA2127⌬HIC(ptsI) as described (12) and purified by ion exchange chromatography over DEAE cellulose (C545, Fluka) and ResourceQ (Amersham Pharmacia) and gel filtration over Superdex 200 (Amersham Pharmacia). DhaK was concentrated to 30 mg⅐ml Ϫ1 in 5 mM Hepes, pH 7.5͞2 mM DTT. The apoform of DhaK was generated between DEAE and ResourceQ chromatography by incubation of the DhaK-Dha complex with PEP and catalytic amounts of the PTS proteins (enzyme I, HPr) DhaM and DhaL. DhaK activity was measured in a coupled assay by reduction of DhaP with glycerol-3-phosphate dehydrogenase. The disappearance of NADH was monitored continuously in a Spectramax 250 plate reader (Molecular Devices) at 30°C (12).Crystallization and Data Collection. The DhaK-Dha complex and the apoprotein were crystallized from 80 mM sodium acetate, pH 5.0͞160 mM (NH 4 ) 2 SO 4 ͞17% (wt͞vol) polyethylene glycol (PEG) 4000͞15% (wt͞vol) 2-methyl-2,4-pentanediol (MPD) using hanging drop vapor diffusion. The crystals had the symmetry of the space group P2 1 2 1 2 (a ϭ 96.5 Å, b ϭ 97.4 Å, c ϭ 86.0 Å, ␣ ϭ ␤ ϭ ␥ ϭ 90°) and they diffracted to 1.75 Å resolution after flash-freezing at 105°K in the native mother liquor. Native and derivative diffraction data were collected on a RAXIS-IV imaging plate detector mounted on a Rigaku RU300 generator equipped with Yale mirrors (Molecular Structure, The Woodlands, TX). All data were processed by using the HKL program package (13).Structure Solution and Refinement. The DhaK structure was determined by the multiple isomorphous replacement (MIR) method. Heavy-atom sites were located with SHELX (14). Phases were calculated by using SHARP (15) and improved by solvent flattening by using the progr...

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

“…The functions of Fak are reminiscent of the analogous Dha kinase components that function in Dha phosphorylation and the control of gene expression (27). E. coli DhaA kinase is composed of three proteins: a phosphotransferase component (DhaM), a nucleotide-binding protein (DhaL), and a Dha-binding protein (DhaK) (28)(29)(30)(31). In C. freundii the DhaL and DhaK are fused to form a functional kinase that uses ATP rather than the phosphotransferase system (6).…”

Regulation of the Dha Operon of Lactococcus lactis

“…Characterization of the DhaS⅐DNA Operator Complex-As shown above DhaS belongs to the TetR family of transcription factors, which employ a helix-turn-helix motif for DNA binding (6). Electrophoretic mobility shift assays confirmed binding of DhaS without a histidine tag to the intergenic region between the dhaS and dhaK genes (Fig.…”

“…The final yield was 60 mg of DhaQ per liter of culture (for details, see supplemental information). The structures of the apo-form of DhaQ and the Dha⅐DhaQ complex were solved by molecular replacement, the apo-form employing the DhaK subunit of E. coli as a search model (6), and the complex using the refined apo-model. Refinement and data collection statistics are given in Table 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).…”

“…Here we present the x-ray structures of the two subunits DhaS and DhaQ and characterize their function. DhaS belongs to the TetR family of transcription regulators (6). But unlike all known members of this family DhaS of L. lactis functions as an activator and not as a repressor of transcription.…”