Literature summary for 3.1.4.45 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.

Cloned(Commentary)

Cloned (Comment)	Organism
polymerase incomplete primer extension cloning method	Homo sapiens
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
C115X	site-directed mutagenesis, the mutation greatly impairs folding of the enzyme, the mutant is retained in the endoplasmic reticulum	Homo sapiens
C132X	site-directed mutagenesis, the mutation greatly impairs folding of the enzyme, the mutant is retained in the endoplasmic reticulum	Homo sapiens
C221L	site-directed mutagenesis, the mutation leads to loss of enzymatic activity	Homo sapiens
C51M	site-directed mutagenesis, the mutant shows 65% of wild-type activity	Homo sapiens
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
N137A	site-directed mutagenesis, the mutant shows 11% of wild-type activity	Homo sapiens
N281A	site-directed mutagenesis, the mutant is recombinantly expressed but retained in the endoplasmic reticulum	Homo sapiens
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
R247A	site-directed mutagenesis, the mutant shows 87% of wild-type activity	Homo sapiens
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
T320A	site-directed mutagenesis, the mutant shows 43% of wild-type activity	Homo sapiens
V318A	site-directed mutagenesis, the mutant is recombinantly expressed but retained in the endoplasmic reticulum	Homo sapiens
V322A	site-directed mutagenesis, the mutant shows 67% of wild-type activity	Homo sapiens

Inhibitors

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

Localization

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

Organism

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

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	-	Homo sapiens

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
6-O-(N-acetyl-alpha-D-glucosaminyl)phospho-D-mannose + H2O	-	Bacteroides ovatus	N-acetyl-D-glucosamine + 6-phospho-D-mannose	-	?
6-O-(N-acetyl-alpha-D-glucosaminyl)phospho-D-mannose + H2O	-	Bacteroides ovatus ATCC 8483	N-acetyl-D-glucosamine + 6-phospho-D-mannose	-	?
6-O-(N-acetyl-alpha-D-glucosaminyl)phospho-D-mannose + H2O	-	Homo sapiens	N-acetyl-D-glucosamine + D-mannose 6-phosphate	-	?
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	-	?
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	-	?
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

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

Synonyms

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

General Information

General Information	Comment	Organism
evolution	the enzyme is a member of the DUF2233-domain protein family	Homo sapiens
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
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
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
physiological function	the enzyme is is involved in targeting of lysosomal enzymes	Homo sapiens