Sidorkina O M, Laval J
Groupe 'Réparation des Lésions Radio- et Chimio-Induites', UMR 1772 CNRS, Institut Gustave Roussy,94805 Villejuif Cédex, France.
Nucleic Acids Res. 1998 Dec 1;26(23):5351-7. doi: 10.1093/nar/26.23.5351.
The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo.
大肠杆菌Fpg蛋白参与氧化残基的修复。我们通过定点诱变研究了第57位保守赖氨酸残基对Fpg蛋白各种催化活性的影响。将赖氨酸57突变为甘氨酸（K57G）的突变型Fpg蛋白切除7,8-二氢-8-氧代鸟嘌呤（8-氧代G）的DNA糖基化酶活性显著降低。野生型Fpg蛋白切割8-氧代G/C DNA的特异性常数（kcat/KM）为0.11/（nM·min），而K57G切割相同DNA的效率低55倍。FpgK57G与8-氧代G/C DNA形成席夫碱复合物的效果很差。K57G突变体与8-氧代G/C DNA双链体的结合效率降低了16倍。将赖氨酸57替换为另一种碱性氨基酸精氨酸（K57R）对8-氧代G-DNA糖基化酶活性和席夫碱形成有轻微影响。以2,6-二氨基-4-羟基-5N-甲基甲酰胺基嘧啶残基为底物时，FpgK57G和FpgK57R的DNA糖基化酶活性与野生型Fpg相当。在体内，与突变体K57R和野生型Fpg相比，突变体K57G仅部分恢复了防止大肠杆菌BH990（fpg mutY）细胞中自发诱导的G/C→T/A转变的能力。这些结果表明赖氨酸57在体外和体内Fpg蛋白的8-氧代G-DNA糖基化酶活性中起重要作用。
