Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene
Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene
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DOI：
10.1128/jb.183.5.1577-1584.2001
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发表时间：
2001-03-01
影响因子：
3.2
通讯作者：
Bobik, TA
中科院分区：
文献类型：
--
作者：
Johnson, CLV;Pechonick, E;Bobik, TA
肠道沙门氏菌通过依赖于辅酶 B-12（腺苷钴胺素 [AdoCb1]）的途径降解 1,2-丙二醇。先前的研究表明，1,2-丙二醇利用 (pdu) 基因包括将无活性的钴胺素（例如维生素 B-12）转化为 AdoCbl 的基因。然而，具体涉及的基因尚未确定。在这里，我们表明 pduO 基因编码具有 ATP:cob(I)alamin 腺苷转移酶活性的蛋白质。该蛋白质的主要作用显然是将无活性的钴胺素转化为 AdoCbl，以降解 1,2-丙二醇。基因测试表明，pduO 基因的功能部分被 cobA 基因（一种已知的 ATP：类咕啉腺苷转移酶）取代，但肠沙门氏菌在 1,2-丙二醇上的最佳生长需要有功能的 pduO 基因。生长研究表明，cobA pduO 双突变体无法在补充有维生素 B-12 的 1,2-丙二醇基本培养基上生长，但能够在补充有 AdoCbl 的类似培养基上生长。 pduO 基因被克隆到 T7 表达载体中。 PduO 蛋白被过表达、部分纯化，并且使用改进的测定程序，显示出具有 cob(I)alamin 腺苷转移酶活性。对编码 PduO 和相关蛋白质的基因的基因组背景的分析表明，特定的腺苷基转移酶往往专门用于特定的 AdoCbl 依赖性酶或 AdoCbl 的从头合成。此类分析还表明 PduO 是一种双功能酶。讨论了与腺苷转移酶同源物邻近的未知功能基因代表先前未鉴定的 AdoCbl 依赖性酶的可能性。
Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B-12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B-12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B-12 but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.