Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea
Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea
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DOI：
10.1007/s00792-007-0094-x
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发表时间：
2007-09-01
期刊：
影响因子：
2.9
通讯作者：
Schoenheit, Peter
中科院分区：
文献类型：
--
作者：
Johnsen, Ulrike;Schoenheit, Peter
磷酸甘油酸酯突变酶（PGM）催化3-磷酸甘油酸酯和2-磷酸甘油酸酯的可逆转化，作为糖酵解和糖异生的一部分。已知两种结构上和机制上不相关的PGM类型，辅因子（2，3-二磷酸甘油酸）依赖性（dPGM）和辅因子非依赖性酶（iPGM）。在这里，我们报告的第一个古细菌辅因子依赖的PGM嗜酸热浆菌，这是由ORF TA 1347编码的表征。该ORF在大肠杆菌中进行了克隆和表达，重组蛋白被鉴定为功能性dPGM。该酶构成一种46 kDa的同二聚体蛋白。酶活性需要2，3-二磷酸甘油酸作为辅因子，并被钒酸盐抑制，钒酸盐是细菌和真核细胞中dPGMs的特异性抑制剂; EDTA可部分缓解抑制作用。T.嗜酸菌，其对应于来自细菌和真核生物的dPGM中的活性位点组氨酸，通过定点诱变被丙氨酸交换。H23 A突变体是催化失活的，支持H23在古细菌dPGM催化中的重要作用。此外，在大肠杆菌中表达后，表征了由来自极端嗜热硫酸盐还原剂闪烁古生球菌的ORF AF 1751编码的古生菌辅因子非依赖性PGM。杆菌单体46 kDa蛋白显示出辅因子非依赖性PGM活性，并被Mn（2+）上标终止物刺激，并在高达70 ℃下表现出高热稳定性。一个全面的系统发育分析这两种类型的古细菌磷酸甘油酸变异酶。
Phosphoglycerate mutases (PGM) catalyze the reversible conversion of 3-phosphoglycerate and 2-phosphoglycerate as part of glycolysis and gluconeogenesis. Two structural and mechanistically unrelated types of PGMs are known, a cofactor (2,3-bisphosphoglycerate)-dependent (dPGM) and a cofactor-independent enzyme (iPGM). Here, we report the characterization of the first archaeal cofactor-dependent PGM from Thermoplasma acidophilum, which is encoded by ORF TA1347. This ORF was cloned and expressed in Escherichia coli and the recombinant protein was characterized as functional dPGM. The enzyme constitutes a 46 kDa homodimeric protein. Enzyme activity required 2,3-bisphosphoglycerate as cofactor and was inhibited by vanadate, a specific inhibitor of dPGMs in bacteria and eukarya; inhibition could be partially relieved by EDTA. Histidine 23 of the archaeal dPGM of T. acidophilum, which corresponds to active site histidine in dPGMs from bacteria and eukarya, was exchanged for alanine by site directed mutagenesis. The H23A mutant was catalytically inactive supporting the essential role of H23 in catalysis of the archaeal dPGM. Further, an archaeal cofactor-independent PGM encoded by ORF AF1751 from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus was characterized after expression in E. coli. The monomeric 46 kDa protein showed cofactor-independent PGM activity and was stimulated by Mn(2+)supercript stop and exhibited high thermostability up to 70 degrees C. A comprehensive phylogenetic analysis of both types of archaeal phosphoglycerate mutases is also presented.