Duwat P, de Oliveira R, Ehrlich S D, Boiteux S
Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Microbiology (Reading). 1995 Feb;141 ( Pt 2):411-7. doi: 10.1099/13500872-141-2-411.
The formamidopyrimidine DNA glycosylase gene (fpg-L) of the Gram-positive microaerophilic bacterium Lactococcus lactis subsp. cremoris ML3 has been cloned, characterized and sequenced. The fpg-L gene is composed of 819 bp encoding a protein of 31.3 kDa (Fpg-L). The deduced amino acid sequence of the Fpg-L protein shows 59% similarity and 38% identity with the Escherichia coli Fpg protein (Fpg-E). Polyclonal antibodies against Fpg-E react with the Fpg-L protein. The Fpg-L protein was purified to apparent homogeneity from the overproducing E. coli strain BH410 hosting plasmid pVE1064, which carries fpg-L under the control of the E. coli lac promoter. In its active form, Fpg-L is a 30 kDa monomeric enzyme with a measured isoelectric point of 9.0. It contains one zinc per molecule and has a zinc finger motif localized at the carboxyterminal end (Cys-X2-Cys-X16-Cys-X2-Cys-X3-COOH). The Fpg-L protein has two enzyme activities: DNA glycosylase, which excises 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine and 7,8-dihydro-8-oxoguanine, and DNA nicking at abasic sites. Furthermore, the expression of the fpg-L gene in fpg and mutY mutants of E. coli suppresses their spontaneous GC-->TA mutator phenotype. The similarity of the activity of the two Fpg proteins and its conversation in evolutionarily distant bacteria may reflect the importance of its role in protecting bacterial DNA against oxidative free radicals.
革兰氏阳性微需氧细菌乳酸乳球菌亚种cremoris ML3的甲酰胺嘧啶DNA糖基化酶基因（fpg-L）已被克隆、表征和测序。fpg-L基因由819个碱基对组成，编码一种31.3 kDa的蛋白质（Fpg-L）。Fpg-L蛋白的推导氨基酸序列与大肠杆菌Fpg蛋白（Fpg-E）显示出59%的相似性和38%的同一性。针对Fpg-E的多克隆抗体与Fpg-L蛋白发生反应。Fpg-L蛋白从携带质粒pVE1064的过量表达大肠杆菌菌株BH410中纯化至表观均一性，该质粒在大肠杆菌lac启动子的控制下携带fpg-L。在其活性形式下，Fpg-L是一种30 kDa的单体酶，测得的等电点为9.0。它每分子含有一个锌，并且在羧基末端有一个锌指基序（Cys-X2-Cys-X16-Cys-X2-Cys-X3-COOH）。Fpg-L蛋白具有两种酶活性：DNA糖基化酶，可切除2,6-二氨基-4-羟基-5N-甲基甲酰胺嘧啶和7,8-二氢-8-氧代鸟嘌呤，以及在无碱基位点进行DNA切口。此外，fpg-L基因在大肠杆菌的fpg和mutY突变体中的表达抑制了它们自发的GC→TA诱变表型。两种Fpg蛋白活性的相似性及其在进化距离较远的细菌中的保守性可能反映了其在保护细菌DNA免受氧化自由基侵害方面作用的重要性。
