EC Number | Cloned (Comment) | Organism |
---|---|---|
2.4.1.83 | recombinant expression of His-tagged wild-type and mutant DELTA230-352 DPMS enzymes in Escherichia coli strain C41(DE3) | Pyrococcus furiosus |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
2.4.1.83 | purified PfDPMS with bound GDP-Man and Mn2+ and with bound GDP and Mg2+, and acceptor substrate Dol55-P, sitting drop vapor diffusion method, 15-20 mg/ml protein is mixed with 5 mM MgCl2 or MnCl2 and either 5 mM GDP or GDP-Man in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% v/v glycerol, and 0.05% LDAO, the protein solution is mixed with crystallization solution containing 0.2 M potassium chloride, 0.1 M trisodium citrate, pH 5.5, and 37% v/v pentaerythritol propoxylate, 4°C, X-ray crystal structure determination and analysis, Dol55-P-Man structure modeling | Pyrococcus furiosus |
2.4.1.83 | structures of DPMS, in complex with nucleotide Mg2+, donor GDP-mannose, and glycolipid product. Substrate binding and product release are orchestrated by an interplay between juxtamembrane interface helices, donor, metal ion and acceptor. Displacement and conformational changes of the juxtamembrane interface helices, most pronouncedly of IFH2, enable entry of the dolichol-phosphate acceptor between IFH1 and IFH2, and docking of its phosphate group at Ser135 (assisted by Arg117 and Arg131), which is located immediately below the mannosyl to be transferred. The acceptor-induced changes in the juxtamembrane interface helices lead to disruption of the A-loop interaction network at the DXD motif and dislodgement of the A-loop. Collapse of the interaction network and opening of the A-loop coincides with concomitant loss of metal ion and attack by the pre-activated nucleophilic dolichol phosphate oxygen on the mannosyl C1 atom | Pyrococcus furiosus |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.4.1.83 | additional information | generation of a PfDPMS truncation variant DELTA230-352 which includes only the catalytic domain | Pyrococcus furiosus |
EC Number | Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|---|
2.4.1.83 | membrane | DPMS is an integral membrane protein | Pyrococcus furiosus | 16020 | - |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
2.4.1.83 | Ca2+ | can substitute for Mg2+ | Pyrococcus furiosus | |
2.4.1.83 | Mg2+ | required, binding structure analysis, overview | Pyrococcus furiosus | |
2.4.1.83 | Mn2+ | can substitute for Mg2+, binding structure analysis, overview | Pyrococcus furiosus |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | Pyrococcus furiosus | - |
GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | Pyrococcus furiosus ATCC 43587 | - |
GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | Pyrococcus furiosus Vc1 | - |
GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | Pyrococcus furiosus JCM 8422 | - |
GDP + dolichyl beta-D-mannosyl phosphate | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.4.1.83 | Pyrococcus furiosus | Q8U4M3 | - |
- |
2.4.1.83 | Pyrococcus furiosus ATCC 43587 | Q8U4M3 | - |
- |
2.4.1.83 | Pyrococcus furiosus JCM 8422 | Q8U4M3 | - |
- |
2.4.1.83 | Pyrococcus furiosus Vc1 | Q8U4M3 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.4.1.83 | recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain C41(DE3) membranes by solubilization with 1% n-dodecyl-beta-D-maltoside in presence of 5% v/v glycerol, nickel affinity chromatography, followed by gel filtration and ultrafiltration. Mutant variant DELTA230-352 is prepared as two fractions, one purified from the membrane fraction (DELTA230-352 m) and one from the aqueous phase (DELTA230-352 s) | Pyrococcus furiosus |
EC Number | Reaction | Comment | Organism | Reaction ID |
---|---|---|---|---|
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate = GDP + dolichyl beta-D-mannosyl phosphate | catalytic mechanism of PfDPMS, overview. Catalytic mechanism of the transfer reaction, nucleophilic attack by the Dol-P phosphate oxygen on the mannosyl C1 carbon yields GDP and Dol-P-Man. Asp91 and Gln93 coordinate the GDP-Man diphosphate groups via the metal ion, while the key side chains for positioning the Dol-P phosphate group for mannosyl transfer are Ser135 and Arg117. The Dol-P phosphate group is pre-activated for nucleophilic attack, and not dependent on a catalytic base for activation | Pyrococcus furiosus |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | - |
Pyrococcus furiosus | GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | GDP-alpha-D-mannose binding structure analysis, overview | Pyrococcus furiosus | GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | - |
Pyrococcus furiosus ATCC 43587 | GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | GDP-alpha-D-mannose binding structure analysis, overview | Pyrococcus furiosus ATCC 43587 | GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | - |
Pyrococcus furiosus Vc1 | GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | GDP-alpha-D-mannose binding structure analysis, overview | Pyrococcus furiosus Vc1 | GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | - |
Pyrococcus furiosus JCM 8422 | GDP + dolichyl beta-D-mannosyl phosphate | - |
? | |
2.4.1.83 | GDP-alpha-D-mannose + dolichyl phosphate | GDP-alpha-D-mannose binding structure analysis, overview | Pyrococcus furiosus JCM 8422 | GDP + dolichyl beta-D-mannosyl phosphate | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.4.1.83 | Dol-PMan synthase | - |
Pyrococcus furiosus |
2.4.1.83 | dolichylphosphate mannose synthase | - |
Pyrococcus furiosus |
2.4.1.83 | DPMS | - |
Pyrococcus furiosus |
2.4.1.83 | PF0058 | - |
Pyrococcus furiosus |
2.4.1.83 | PfDPMS | - |
Pyrococcus furiosus |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
2.4.1.83 | 75 | - |
assay at | Pyrococcus furiosus |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
2.4.1.83 | 7.5 | - |
assay at | Pyrococcus furiosus |
EC Number | General Information | Comment | Organism |
---|---|---|---|
2.4.1.83 | evolution | PfDPMS is a membrane enzymes of the GT2 family. All known bona fide DPMSs use GDP-Man as donor substrate. Proposed model of substrate binding and product release | Pyrococcus furiosus |
2.4.1.83 | malfunction | failure to produce or utilize dolichyl mannosyl phosphate compromises organism viability | Pyrococcus furiosus |
2.4.1.83 | metabolism | the enzyme is involved in dolichylphosphate mannose biosynthesis | Pyrococcus furiosus |
2.4.1.83 | additional information | structure-function analysis, and structural comparison with related enzymes, detailed overview. The IF helices IFH1 and IFH2 are important for activity | Pyrococcus furiosus |
2.4.1.83 | physiological function | the membrane protein dolichylphosphate mannose synthase (DPMS) catalyzes the reaction whereby mannose is transferred from GDP-mannose to the dolichol carrier dolichyl phosphate (Dol-P), to yield dolichyl mannosyl phosphate (Dol-P-Man). Lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol-P-Man synthesis | Pyrococcus furiosus |