title: Molecular characterization of the human Calpha-formylglycine-generating enzyme.
abstract: Calpha-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calcium-binding protein containing an N-terminal (residues 86-168) and a C-terminal (residues 178-374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the N-terminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocross-linking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.
authors.affiliation: Institut für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Preusser-Kunze
authors.forename: Malaiyalam
authors.initials: M
authors.lastname: Mariappan
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schmidt
authors.forename: Santosh Lakshmi
authors.initials: SL
authors.lastname: Gande
authors.forename: Kudzai
authors.initials: K
authors.lastname: Mutenda
authors.forename: Dirk
authors.initials: D
authors.lastname: Wenzel
authors.forename: Kurt
authors.initials: K
authors.lastname: von Figura
authors.forename: Thomas
authors.initials: T
authors.lastname: Dierks
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013429:Sulfatases / Q000737:chemistry*; D013820:Thermolysin / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
chemical_list: D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D010455:Peptides; D011134:Polysaccharides; D011994:Recombinant Proteins; D011392:Proline; D050862:Oxidoreductases Acting on Sulfur Group Donors; C480483:SUMF1 protein, human; D013429:Sulfatases; D006026:Glycoside Hydrolases; D014357:Trypsin; D013820:Thermolysin; D003545:Cysteine; D005033:Ethylmaleimide