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Literature summary extracted from

  • Das, D.; Lee, W.S.; Grant, J.C.; Chiu, H.J.; Farr, C.L.; Vance, J.; Klock, H.E.; Knuth, M.W.; Miller, M.D.; Elsliger, M.A.; Deacon, A.M.; Godzik, A.; Lesley, S.A.; Kornfeld, S.; Wilson, I.A.
    Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme (2013), J. Biol. Chem., 288, 16789-16799.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.1.4.45 polymerase incomplete primer extension cloning method Homo sapiens
3.1.4.45 polymerase incomplete primer extension cloning method, recombinant expression of enzyme with TEV cleavage site and His6-tag in Escherichia coli as wild-type and selenomethionine-labeled enzyme Bacteroides ovatus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.1.4.45 purified recombinant detagged wild-type and selenomethionine-labeled enzyme, sitting nanodroplet vapor diffusion method, mixing of 200 nl of 21 mg/ml protein in 20 mM HEPES, pH 8.0, 200 mM NaCl, 40 mM imidazole, and 1 mM TCEP, with 200 nl of reservoir solution containing 0.2 M Li2SO4, 30% PEG 4000, and 0.1 M Tris, pH 8.5, and equilibration against 0.05 ml of reservoir solution, 4°C, 10% v/v ethylene glycol as a cryoprotectant, X-ray diffraction structure determination and analysis at 1.80 A resolution, modeling, the crystal structure of the BACOVA_00430 protein from Bacteroides ovatus is used to model the luminal portion of the human enzyme N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase Bacteroides ovatus

Protein Variants

EC Number Protein Variants Comment Organism
3.1.4.45 C115X site-directed mutagenesis, the mutation greatly impairs folding of the enzyme, the mutant is retained in the endoplasmic reticulum Homo sapiens
3.1.4.45 C132X site-directed mutagenesis, the mutation greatly impairs folding of the enzyme, the mutant is retained in the endoplasmic reticulum Homo sapiens
3.1.4.45 C221L site-directed mutagenesis, the mutation leads to loss of enzymatic activity Homo sapiens
3.1.4.45 C51M site-directed mutagenesis, the mutant shows 65% of wild-type activity Homo sapiens
3.1.4.45 C51M/C221L site-directed mutagenesis, the mutant folds adequately and is trafficked to the Golgi where it is cleaved by furin, the mutant shows only 9.7% of wild-type activity Homo sapiens
3.1.4.45 N137A site-directed mutagenesis, the mutant shows 11% of wild-type activity Homo sapiens
3.1.4.45 N281A site-directed mutagenesis, the mutant is recombinantly expressed but retained in the endoplasmic reticulum Homo sapiens
3.1.4.45 Q225H site-directed mutagenesis, exchange of the human residue for the residue of protein BACOVA_00430 from Bacteroides ovatus, the mutant shows 5.8% of wild-type activity Homo sapiens
3.1.4.45 R247A site-directed mutagenesis, the mutant shows 87% of wild-type activity Homo sapiens
3.1.4.45 T227R site-directed mutagenesis, exchange of the human residue for the residue of protein BACOVA_00430 from Bacteroides ovatus, the mutant shows 0.1% of wild-type activity Homo sapiens
3.1.4.45 T320A site-directed mutagenesis, the mutant shows 43% of wild-type activity Homo sapiens
3.1.4.45 V318A site-directed mutagenesis, the mutant is recombinantly expressed but retained in the endoplasmic reticulum Homo sapiens
3.1.4.45 V322A site-directed mutagenesis, the mutant shows 67% of wild-type activity Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.1.4.45 D-glucosamine 6-phosphate
-
 Bacteroides ovatus
3.1.4.45 D-glucosamine 6-phosphate
-
 Homo sapiens
3.1.4.45 D-glucose 1-phosphate
-
 Bacteroides ovatus
3.1.4.45 D-glucose 1-phosphate
-
 Homo sapiens
3.1.4.45 D-glucose 6-phosphate
-
 Bacteroides ovatus
3.1.4.45 D-glucose 6-phosphate
-
 Homo sapiens
3.1.4.45 D-mannose 1-phosphate
-
 Bacteroides ovatus
3.1.4.45 D-mannose 1-phosphate
-
 Homo sapiens
3.1.4.45 D-mannose 6-phosphate
-
 Bacteroides ovatus
3.1.4.45 D-mannose 6-phosphate
-
 Homo sapiens
3.1.4.45 GDP-mannose
-
 Bacteroides ovatus
3.1.4.45 GDP-mannose
-
 Homo sapiens
3.1.4.45 N-acetyl-L-galactosamine
-
 Bacteroides ovatus
3.1.4.45 N-acetyl-L-galactosamine
-
 Homo sapiens
3.1.4.45 N-acetyl-L-glucosamine
-
 Bacteroides ovatus
3.1.4.45 N-acetyl-L-glucosamine
-
 Homo sapiens
3.1.4.45 N-acetyl-L-glucosamine 1-phosphate
-
 Bacteroides ovatus
3.1.4.45 N-acetyl-L-glucosamine 1-phosphate
-
 Homo sapiens
3.1.4.45 N-acetyl-L-mannosamine
-
 Bacteroides ovatus
3.1.4.45 N-acetyl-L-mannosamine
-
 Homo sapiens
3.1.4.45 phosphate
-
 Bacteroides ovatus
3.1.4.45 phosphate
-
 Homo sapiens
3.1.4.45 UDP-galactose
-
 Bacteroides ovatus
3.1.4.45 UDP-galactose
-
 Homo sapiens
3.1.4.45 UDP-glucose
-
 Bacteroides ovatus
3.1.4.45 UDP-glucose
-
 Homo sapiens
3.1.4.45 UDP-N-acetyl-galactosamine
-
 Bacteroides ovatus
3.1.4.45 UDP-N-acetyl-galactosamine
-
 Homo sapiens
3.1.4.45 UDP-N-acetyl-glucosamine
-
 Bacteroides ovatus
3.1.4.45 UDP-N-acetyl-glucosamine
-
 Homo sapiens

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.1.4.45 Golgi membrane a transmembrane enzyme Homo sapiens 139
-

Organism

EC Number Organism UniProt Comment Textmining
3.1.4.45 Bacteroides ovatus A7LRK2 a human gut bacterium
-
3.1.4.45 Bacteroides ovatus ATCC 8483 A7LRK2 a human gut bacterium
-
3.1.4.45 Homo sapiens
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.1.4.45 glycoprotein
-
 Homo sapiens

Purification (Commentary)

EC Number Purification (Comment) Organism
3.1.4.45 recombinant enzyme with TEV cleavage site and His6-tag by nickel affinity chromatography, tag cleavage by tobacco etch virus protease, another step of nickel affinity chromatography, and centrifugal ultrafiltration Bacteroides ovatus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.4.45 6-O-(N-acetyl-alpha-D-glucosaminyl)phospho-D-mannose + H2O
-
 Bacteroides ovatus N-acetyl-D-glucosamine + 6-phospho-D-mannose
-
 ?
3.1.4.45 6-O-(N-acetyl-alpha-D-glucosaminyl)phospho-D-mannose + H2O
-
 Bacteroides ovatus ATCC 8483 N-acetyl-D-glucosamine + 6-phospho-D-mannose
-
 ?
3.1.4.45 6-O-(N-acetyl-alpha-D-glucosaminyl)phospho-D-mannose + H2O
-
 Homo sapiens N-acetyl-D-glucosamine + D-mannose 6-phosphate
-
 ?
3.1.4.45 methyl 6-O-(N-acetyl-alpha-D-glucosaminyl)phospho-alpha-D-mannose + H2O low activity Homo sapiens N-acetyl-D-glucosamine + methyl alpha-D-mannose 6-phosphate
-
 ?
3.1.4.45 methyl 6-O-(N-acetyl-D-glucosaminyl)phospho-alpha D-mannose + H2O low activity Bacteroides ovatus N-acetyl-D-glucosamine + methyl 6-phospho-alpha-D-mannopyranoside
-
 ?
3.1.4.45 methyl 6-O-(N-acetyl-D-glucosaminyl)phospho-alpha D-mannose + H2O low activity Bacteroides ovatus ATCC 8483 N-acetyl-D-glucosamine + methyl 6-phospho-alpha-D-mannopyranoside
-
 ?

Subunits

EC Number Subunits Comment Organism
3.1.4.45 monomer SDS-PAGE and gel filtration Bacteroides ovatus

Synonyms

EC Number Synonyms Comment Organism
3.1.4.45 BACOVA_00430
-
 Bacteroides ovatus
3.1.4.45 UCE
-
 Homo sapiens
3.1.4.45 uncovering enzyme
-
 Homo sapiens

General Information

EC Number General Information Comment Organism
3.1.4.45 evolution the enzyme is a member of the DUF2233-domain protein family Homo sapiens
3.1.4.45 evolution the enzyme is a member of the DUF2233-domain protein family. The most conserved residues are Asn130, His217, Arg219, Arg239, Asn268, Asp270, Gly271, Gly272, Gly273, Ser274, Arg303, and Val305. These residues are clustered on one side of the protein, and almost all have significant surface exposure. Of these, Asn268, Asp270, Gly271-273, and Ser274 are part of the highly conserved A(I/L)NLDGGGS(T/S/A)T motif present throughout the DUF2233 family and located in helix H7 and in the preceding loop between beta17 and H7 near the center of the conserved site Bacteroides ovatus
3.1.4.45 additional information a sulfate ion is bound near Gly272-Gly273 of the GGGS sequence and anchored by conserved residues Arg239 and Arg303 and may represent the binding site for the phosphate moiety of the substrate. Asn130, Asp270, and Ser274 are potential catalytic residues Bacteroides ovatus
3.1.4.45 additional information the crystal structure of the BACOVA_00430 protein from Bacteroides ovatus is used to model the luminal portion of human enzyme. The Cys51-Cys221 disulfide bond is not absolutely essential for folding or enzyme activity Homo sapiens
3.1.4.45 physiological function the enzyme is is involved in targeting of lysosomal enzymes Homo sapiens