Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases.
Sulfatases carry at their catalytic site a unique post-translational modification, an alpha-formylglycine residue that is essential for enzyme activity. Formylglycine is generated by oxidation of a conserved cysteine or, in some prokaryotic sulfatases, serine residue. In eukaryotes, this oxidation occurs in the endoplasmic reticulum during or shortly after import of the nascent sulfatase polypeptide. The modification of arylsulfatase A was studied in vitro and was found to be directed by a short linear sequence, CTPSR, starting with the cysteine to be modified. Mutational analyses showed that the cysteine, proline and arginine are the key residues within this motif, whereas formylglycine formation tolerated the individual, but not the simultaneous substitution of the threonine or serine. The CTPSR motif was transferred to a heterologous protein leading to low-efficient formylglycine formation. The efficiency reached control values when seven additional residues (AALLTGR) directly following the CTPSR motif in arylsulfatase A were present. Mutating up to four residues simultaneously within this heptamer sequence inhibited the modification only moderately. AALLTGR may, therefore, have an auxiliary function in presenting the core motif to the modifying enzyme. Within the two motifs, the key residues are fully, and other residues are highly conserved among all known members of the sulfatase family.
Dierks
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Institut für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Gosslerstrasse 12d, D-37073 Göttingen, Germany. dierks@ukb2-00.uni-bc.gwdg.de
Lecca
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MR
Schlotterhose
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P
Schmidt
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B
von Figura
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D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002553:Cerebroside-Sulfatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; C524739:C(alpha)-formylglycine; D002553:Cerebroside-Sulfatase; D003545:Cysteine; D000409:Alanine; D005998:Glycine