用黄素和黄素类似物对大肠杆菌光解酶进行重组。

Reconstitution of Escherichia coli photolyase with flavins and flavin analogues.

作者信息

Payne G, Wills M, Walsh C, Sancar A

机构信息

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.

出版信息

Biochemistry. 1990 Jun 19;29(24):5706-11. doi: 10.1021/bi00476a010.

DOI:10.1021/bi00476a010

PMID:2200512

Abstract

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.

摘要

大肠杆菌DNA光解酶含有两种发色团辅因子，即1,5-二氢黄素腺嘌呤二核苷酸（FADH2）和（5,10-亚甲基四氢叶酸）聚谷氨酸（5,10-MTHF）。已开发出一种用于脱辅基光解酶及其发色团可逆拆分的方法。为了研究对于FAD与脱辅基光解酶结合以及光解酶与DNA结合至关重要的结构，尝试了用FAD、FMN、核黄素、1-脱氮FAD、5-脱氮FAD和F420进行重组实验。只有FAD和5-脱氮FAD显示出与脱辅基光解酶的高亲和力结合。脱辅基酶对DNA没有亲和力，但在化学计量结合FAD或5-脱氮FAD后，确实恢复了其与含胸腺嘧啶二聚体的DNA的特异性结合。用连二亚硫酸盐成功还原酶结合的FAD导致光催化活性完全恢复。