Dierks T, Miech C, Hummerjohann J, Schmidt B, Kertesz M A, von Figura K
Institut für Biochemie und Molekulare Zellbiologie, Abt. Biochemie II, Universität Göttingen, Gosslerstrasse 12d, 37073 Göttingen, Germany.
J Biol Chem. 1998 Oct 2;273(40):25560-4. doi: 10.1074/jbc.273.40.25560.
Eukaryotic sulfatases carry an alpha-formylglycine residue that is essential for activity and is located within the catalytic site. This formylglycine is generated by posttranslational modification of a conserved cysteine residue. The arylsulfatase gene of Pseudomonas aeruginosa also encodes a cysteine at the critical position. This protein could be expressed in active form in a sulfatase-deficient strain of P. aeruginosa, thereby restoring growth on aromatic sulfates as sole sulfur source, and in Escherichia coli. Analysis of the mature protein expressed in E. coli revealed the presence of formylglycine at the expected position, showing that the cysteine is also converted to formylglycine in a prokaryotic sulfatase. Substituting the relevant cysteine by a serine codon in the P. aeruginosa gene led to expression of inactive sulfatase protein, lacking the formylglycine. The machinery catalyzing the modification of the Pseudomonas sulfatase in E. coli therefore resembles the eukaryotic machinery, accepting cysteine but not serine as a modification substrate. By contrast, in the arylsulfatase of Klebsiella pneumoniae a formylglycine is found generated by modification of a serine residue. The expression of both the Klebsiella and the Pseudomonas sulfatases as active enzymes in E. coli suggests that two modification systems are present, or that a common modification system is modulated by a cofactor.
真核生物硫酸酯酶带有一个对活性至关重要的α-甲酰甘氨酸残基，该残基位于催化位点内。这种甲酰甘氨酸是由一个保守的半胱氨酸残基经翻译后修饰产生的。铜绿假单胞菌的芳基硫酸酯酶基因在关键位置也编码一个半胱氨酸。该蛋白可以在铜绿假单胞菌的硫酸酯酶缺陷菌株中以活性形式表达，从而恢复以芳香族硫酸盐作为唯一硫源时的生长，并且也能在大肠杆菌中表达。对在大肠杆菌中表达的成熟蛋白进行分析发现，在预期位置存在甲酰甘氨酸，这表明在原核生物硫酸酯酶中半胱氨酸也会转化为甲酰甘氨酸。在铜绿假单胞菌基因中用丝氨酸密码子替换相关半胱氨酸会导致无活性的硫酸酯酶蛋白表达，该蛋白缺乏甲酰甘氨酸。因此，在大肠杆菌中催化铜绿假单胞菌硫酸酯酶修饰的机制类似于真核生物机制，接受半胱氨酸而非丝氨酸作为修饰底物。相比之下，在肺炎克雷伯菌的芳基硫酸酯酶中，甲酰甘氨酸是由丝氨酸残基修饰产生的。肺炎克雷伯菌和铜绿假单胞菌的硫酸酯酶在大肠杆菌中均以活性酶形式表达，这表明存在两种修饰系统，或者一种共同的修饰系统受辅因子调节。
