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Literature summary extracted from

  • Gandini, R.; Reichenbach, T.; Tan, T.; Divne, C.
    Structural basis for dolichylphosphate mannose biosynthesis (2017), Nat. Commun., 8, 120 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

Crystallization (Commentary)

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

Protein Variants

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

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.4.1.83 membrane DPMS is an integral membrane protein Pyrococcus furiosus 16020
-

Metals/Ions

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

Natural Substrates/ Products (Substrates)

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
-
?

Organism

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
-
-

Purification (Commentary)

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

Reaction

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

Substrates and Products (Substrate)

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
-
?

Synonyms

EC Number Synonyms Comment Organism
2.4.1.83 Dol-PMan synthase
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Pyrococcus furiosus
2.4.1.83 dolichylphosphate mannose synthase
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Pyrococcus furiosus
2.4.1.83 DPMS
-
Pyrococcus furiosus
2.4.1.83 PF0058
-
Pyrococcus furiosus
2.4.1.83 PfDPMS
-
Pyrococcus furiosus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.83 75
-
assay at Pyrococcus furiosus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.83 7.5
-
assay at Pyrococcus furiosus

General Information

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