The bifunctional protein hldE includes D-glycero-beta-D-manno-heptose-7-phosphate kinase and D-glycero-beta-D-manno-heptose 1-phosphate adenylyltransferase activity (cf. EC 2.7.1.167). The enzyme is involved in biosynthesis of ADP-L-glycero-beta-D-manno-heptose, which is utilized for assembly of the lipopolysaccharide inner core in Gram-negative bacteria.
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The bifunctional protein hldE includes D-glycero-beta-D-manno-heptose-7-phosphate kinase and D-glycero-beta-D-manno-heptose 1-phosphate adenylyltransferase activity (cf. EC 2.7.1.167). The enzyme is involved in biosynthesis of ADP-L-glycero-beta-D-manno-heptose, which is utilized for assembly of the lipopolysaccharide inner core in Gram-negative bacteria.
rfaE encodes a bifunctional protein. It is proposed that domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose
bifunctional D-beta-D-heptose-7-phosphate kinase/D-beta-dheptose-1-phosphate adenylyltransferase. Based on genomic sequence comparisons, bifunctional proteins are predicted to be present in several Gram-negative microorganisms, including Agrobacterium tumefaciens, Buchnera sp., Caulobacter crescentus, Salmonella typhimurium, Salmonella typhi, Vibrio cholerae, Yersinia pestis, Haemophilus influenzae, Helicobacter pylori and Pseudomonas aeruginosa. In contrast, individual genes encoding domains I and II independently are found in Ralstonia eutropha, Neisseria meningitidis and Neisseria gonorrhoeae. In these cases, it is proposed to use the nomenclature hldA and hldC to indicate the individual kinase- and adenylyltransferase-encoding genes, respectively
rfaE encodes a bifunctional protein. It is proposed that domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose
BpHldC belongs to the nucleotidyltransferase alpha/beta phosphodiesterase superfamily sharing a common Rossmann-like alpha/beta fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues
BpHldC belongs to the nucleotidyltransferase alpha/beta phosphodiesterase superfamily sharing a common Rossmann-like alpha/beta fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues
D-glycero-beta-D-manno-heptose-1-phosphate adenylyltransferase (HldC) is the fourth enzyme of the ADP-1-glycero-beta-D-manno-heptose biosynthesis pathway, which produces an essential carbohydrate comprising the inner core of lipopolysaccharide
D-glycero-beta-D-manno-heptose-1-phosphate adenylyltransferase (HldC) is the fourth enzyme of the ADP-1-glycero-beta-D-manno-heptose biosynthesis pathway, which produces an essential carbohydrate comprising the inner core of lipopolysaccharide
the crystal structure of BpHldC belongs to the nucleotidyltransferase alpha/beta phosphodiesterase superfamily sharing a common Rossmann-like alpha/beta fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues. Intriguingly, a reorientation of the C-terminal helix seems to guide open and close states of the active site for the catalytic reaction. Active site structure analysis and structure comparisons, overview. Catalytic mechanism is inferred from the sequence and structure analysis: general, the first requirement of the nucleotidyl transfer reaction in the superfamily is to bring NTP and substrate in the proper orientation. The residues on the T/HXGH motif, flap domain and initial portion of alpha7 helix accomplish the condition by making interaction with the phosphate moieties of NTP and stabilizing the highly charged pentacovalent transition state. The catalytic activity of BpHldC may also require an adequate position of b-H1P in the correct orientation. Then, the phosphate moiety of beta-H1P undergoes nucleophilic attack on the alpha-phosphate of the ATP in an in-line displacement mechanism. The formation of the stable transition state by the key basic residues (His 40, Lys69) is presumed to be essential in the catalytic mechanism as shown in other family members
the crystal structure of BpHldC belongs to the nucleotidyltransferase alpha/beta phosphodiesterase superfamily sharing a common Rossmann-like alpha/beta fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues. Intriguingly, a reorientation of the C-terminal helix seems to guide open and close states of the active site for the catalytic reaction. Active site structure analysis and structure comparisons, overview. Catalytic mechanism is inferred from the sequence and structure analysis: general, the first requirement of the nucleotidyl transfer reaction in the superfamily is to bring NTP and substrate in the proper orientation. The residues on the T/HXGH motif, flap domain and initial portion of alpha7 helix accomplish the condition by making interaction with the phosphate moieties of NTP and stabilizing the highly charged pentacovalent transition state. The catalytic activity of BpHldC may also require an adequate position of b-H1P in the correct orientation. Then, the phosphate moiety of beta-H1P undergoes nucleophilic attack on the alpha-phosphate of the ATP in an in-line displacement mechanism. The formation of the stable transition state by the key basic residues (His 40, Lys69) is presumed to be essential in the catalytic mechanism as shown in other family members
the N-terminal domain I spans amino acids 1-318 and shares structural features with members of the ribokinase family. The C-terminal domain II, which spans amino acids 344-477, has all the conserved features of the cytidylyltransferase superfamily
the wild-type overall architecture belongs to a three layer (alpha/beta/alpha) sandwich structure with a central beta-sheet topology followed by a C-terminal helix through a flexible loop. There are four protomers in the unit cell and all of them exist in a dimeric form. The different contacts of two dimeric pairs are presumed to be originated from different crystal packing environment. It implies inherent flexibility of the hinge loop connecting the C-terminal helix. The N-terminal domain of BpHldC is attached to the main catalytic domain by forming hydrophobic core consisting of Leu15, Ile55, Val95, Leu105, and Val109. The C-terminal helix domain seems to be connected to the main catalytic domain by a hinge loop in the superfamily
the wild-type overall architecture belongs to a three layer (alpha/beta/alpha) sandwich structure with a central beta-sheet topology followed by a C-terminal helix through a flexible loop. There are four protomers in the unit cell and all of them exist in a dimeric form. The different contacts of two dimeric pairs are presumed to be originated from different crystal packing environment. It implies inherent flexibility of the hinge loop connecting the C-terminal helix. The N-terminal domain of BpHldC is attached to the main catalytic domain by forming hydrophobic core consisting of Leu15, Ile55, Val95, Leu105, and Val109. The C-terminal helix domain seems to be connected to the main catalytic domain by a hinge loop in the superfamily
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified SeMet-labeled and wild-type His6-tagged enzymes are crystallized from 0.1 M HEPES, pH 7.0, 20% w/v PEG 3350, 8.5 mM n-octyl-beta-D-thiomaltoside, and from 0.1 M MES, pH 6.5, 15% w/v PEG 3350, and 6-cyclohexyl-L-hexyl-beta-D-maltoside, respectively, hanging drop vapour diffusion method, X-ray diffraction structure determination and analysis at 2.80 A and 2.40 A resolution, respectively, modeling, the refined model of SeMet-BpHldC is used as a search model for molecular replacement (MR) to solve the structure of native BpHldC
purified SeMet-labeled enzyme containing a noncleavable N-terminal His6-tag, hanging drop vapour diffusion method, mixing of 800 nl of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 0.35 M NaCl with 800 nl of reservoir solution containing 0.1 M HEPES, pH 7.0, 20% w/v PEG 3350, and 8.5 mM n-octyl-beta-D-thiomaltoside, and equilibration over 0.05 ml of reservoir solution, room temperature, method optimization, X-ray diffraction structure determination and analysis at 2.80 A resolution, rod-shaped crystals, automated model building
recombinant N-terminally His6-tagged SeMet-labeled BpHldC from Escherichia coli strain B834(DE3) by nickel affinity and anion exchange chromatography to about 99% purity
Valvano, M.A.; Marolda, C.L.; Bittner, M.; Glaskin-Clay, M.; Simon, T.L.; Klena, J.D.
The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose