Literature summary for 2.4.1.83 extracted from

Gandini, R.; Reichenbach, T.; Tan, T.; Divne, C.
Structural basis for dolichylphosphate mannose biosynthesis (2017), Nat. Commun., 8, 120 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant DELTA230-352 DPMS enzymes in Escherichia coli strain C41(DE3)	Pyrococcus furiosus

Crystallization (Commentary)

Crystallization (Comment)	Organism
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
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

Crystallization (Comment)

Organism

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

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

Protein Variants	Comment	Organism
additional information	generation of a PfDPMS truncation variant DELTA230-352 which includes only the catalytic domain	Pyrococcus furiosus

Localization

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

Metals/Ions

Metals/Ions	Comment	Organism
Ca2+	can substitute for Mg2+	Pyrococcus furiosus
Mg2+	required, binding structure analysis, overview	Pyrococcus furiosus
Mn2+	can substitute for Mg2+, binding structure analysis, overview	Pyrococcus furiosus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
GDP-alpha-D-mannose + dolichyl phosphate	Pyrococcus furiosus	-	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	Pyrococcus furiosus ATCC 43587	-	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	Pyrococcus furiosus Vc1	-	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	Pyrococcus furiosus JCM 8422	-	GDP + dolichyl beta-D-mannosyl phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus furiosus	Q8U4M3	-	-
Pyrococcus furiosus ATCC 43587	Q8U4M3	-	-
Pyrococcus furiosus JCM 8422	Q8U4M3	-	-
Pyrococcus furiosus Vc1	Q8U4M3	-	-

Purification (Commentary)

Purification (Comment)	Organism
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

Purification (Comment)

Organism

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

Reaction	Comment	Organism	Reaction ID
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	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
GDP-alpha-D-mannose + dolichyl phosphate	-	Pyrococcus furiosus	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	GDP-alpha-D-mannose binding structure analysis, overview	Pyrococcus furiosus	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	-	Pyrococcus furiosus ATCC 43587	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	GDP-alpha-D-mannose binding structure analysis, overview	Pyrococcus furiosus ATCC 43587	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	-	Pyrococcus furiosus Vc1	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	GDP-alpha-D-mannose binding structure analysis, overview	Pyrococcus furiosus Vc1	GDP + dolichyl beta-D-mannosyl phosphate	-	?
GDP-alpha-D-mannose + dolichyl phosphate	-	Pyrococcus furiosus JCM 8422	GDP + dolichyl beta-D-mannosyl phosphate	-	?
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

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

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information	Comment	Organism
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
malfunction	failure to produce or utilize dolichyl mannosyl phosphate compromises organism viability	Pyrococcus furiosus
metabolism	the enzyme is involved in dolichylphosphate mannose biosynthesis	Pyrococcus furiosus
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
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