Jolley Keith A, Maddocks Deborah G, Gyles Shan L, Mullan Zoë, Tang Sen-Lin, Dyall-Smith Michael L, Hough David W, Danson Michael J
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK1.
Department of Microbiology & Immunology, University of Melbourne, Parkville, Australia2.
Microbiology (Reading). 2000 May;146 ( Pt 5):1061-1069. doi: 10.1099/00221287-146-5-1061.
All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase. This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase multienzyme complex [PDHC], consisting of multiple copies of three component enzymes: E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH). Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined. In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported. Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1alpha, E1beta and E2 genes of the PDHC from gram-positive organisms. Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved. Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts. The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent multienzyme complex assembly. This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed.
所有古生菌都通过一种简单的丙酮酸氧化还原酶催化丙酮酸转化为乙酰辅酶A。这与真核生物和大多数需氧细菌不同，真核生物和大多数需氧细菌使用由三种组成酶（E1，丙酮酸脱羧酶；E2，硫辛酰乙酰转移酶；E3，二氢硫辛酰胺脱氢酶，DHLipDH）的多个拷贝组成的丙酮酸脱氢酶多酶复合体[PDHC]。到目前为止，在古生菌中尚未发现PDHC活性，尽管在极端嗜盐古生菌中发现了DHLipDH，并且其基因序列已被确定。本文报道了嗜盐古生菌沃氏嗜盐菌中一个包含DHLipDH基因的操纵子的发现和测序。在DHLipDH基因上游有3个开放阅读框，它们与革兰氏阳性生物的PDHC的E1α、E1β和E2基因具有最高的序列同一性。对E2基因推测的蛋白质产物的结构预测显示出PDHC中E2组分的结构域特征，并且包括硫辛酸辅因子共价结合的赖氨酸在内的催化重要残基是保守的。Northern分析表明整个操纵子都有转录，但在细胞提取物中未检测到PDHC酶活性。在E2基因中存在细菌或真核生物E2蛋白中未发现的插入片段（约相当于100个氨基酸），可能预计会阻止多酶复合体的组装。这是古生菌中一个假定的2-氧代酸脱氢酶复合体基因的首次详细报道，并讨论了这些发现的进化和代谢后果。
