Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium
Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium
复制标题
DOI：
10.1042/bj20021443
复制
发表时间：
2003-03-01
影响因子：
4.1
通讯作者：
Warren, MJ
中科院分区：
文献类型：
--
作者：
Raux, E;Leech, HK;Warren, MJ
在巨大芽孢杆菌中，分离出了 hemAXBCDL 基因，发现其与枯草芽孢杆菌中的基因高度相似，而枯草芽孢杆菌是将谷氨酰-tRNA 转化为尿卟啉原 III 所需的基因。 HemC（胆色素原脱氨酶）和 -D（尿卟啉原 III 合酶）的过量生产和纯化使得这些酶可用于从胆色素原体外合成尿卟啉原 III。第二个较小的三个基因簇（称为 SirABC）也被分离出来，并被发现编码催化尿卟啉原 III 转化为西罗血红素的酶，因为它们能够补充特定的大肠杆菌 (cysG) 突变体。通过直接酶测定研究了 SirC 和 -B 的功能，其中发现 SirC 充当前 Corrin-2 脱氢酶，产生西罗氢氯酸，而 SirB 被发现充当亚铁螯合酶，负责西罗血红素合成的最后步骤。 CbiX 是一种在巨大芽孢杆菌主要钴胺素生物合成操纵子中编码的蛋白质，与 SirB 具有高度相似性，并且通过将钴插入西罗氢氯酸中，充当与钴胺素生物合成相关的钴螯合酶。 CbiX 在蛋白质的 C 末端部分包含所有不寻常的富含组氨酸的区域，但未发现该区域在螯合过程中是必需的。序列比对表明 SirB 和 CbiX 与肠沙门氏菌的钴螯合酶 CbiK 具有相似的活性位点。
In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin- 2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains all unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.