Sequence determinants directing conversion of cysteine to formylglycine ineukaryotic sulfatases

Sulfatases carry at their catalytic site a unique posttranslational modific ation, an alpha-formylglycine residue that is essential for enzyme activity . Formylglycine is generated by oxidation of a conserved cysteine or, in so me prokaryotic sulfatases, serine residue. In eukaryotes, this oxidation oc curs in the endoplasmic reticulum during or shortly after import of the nas cent sulfatase polypeptide, The modification of arylsulfatase A was studied in vitro and was found to be directed by a short linear sequence, CTPSR, s tarting with the cysteine to be modified. Mutational analyses showed that t he cysteine, proline and arginine are the key residues within this motif, w hereas formylglycine formation tolerated the individual, but not the simult aneous substitution of the threonine or serine. The CTPSR moth was transfer red to a heterologous protein leading to low-efficient formylglycine format ion. The efficiency reached control values when seven additional residues ( AALLTGR) directly following the CTPSR moth 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. W ithin the two moths, the key residues are fully, and other residues are hig hly conserved among all known members of the sulfatase family.