Sielaff Bernhard, Andreesen Jan R
Institut für Mikrobiologie, Martin-Luther-Universität Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
Microbiology (Reading). 2005 Aug;151(Pt 8):2593-2603. doi: 10.1099/mic.0.28039-0.
Cloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450(mor)) and the Fe(3)S(4) ferredoxin (Fd(mor)), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450(mor) system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf1', encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR(mor) was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K(m) values of FdR(mor) for the substrate NADH (37.7 +/- 4.1 microM) and the artificial electron acceptors potassium ferricyanide (14.2 +/- 1.1 microM) and cytochrome c (28.0 +/- 3.6 microM) were measured. FdR(mor) was shown to interact functionally with its natural redox partner, the Fe(3)S(4) protein Fd(mor), and with the Fe(2)S(2) protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd(mor) in activity assays. These results indicated that FdR(mor) can utilize different ferredoxins, but that Fd(mor) requires the specific NADH : ferredoxin oxidoreductase FdR(mor) from the P450(mor) system for efficient catalytic function.
morABC操纵子区域的克隆和测序揭示了编码细胞色素P450单加氧酶三个组分的基因，该酶是分枝杆菌属菌株HE5降解N-杂环吗啉所必需的。分别由morA和morB编码的细胞色素P450（P450(mor)）和Fe(3)S(4)铁氧化还原蛋白（Fd(mor)）先前已得到表征，然而，迄今为止尚未获得支持P450(mor)系统活性所需的特异性吗啉诱导型还原酶的证据。对mor操纵子的分析现已揭示了morC基因，其编码这种吗啉单加氧酶的铁氧化还原蛋白还原酶。morA、morB和morC基因与分枝杆菌属菌株RP1的相应基因相同。现在已在嗜氯分枝杆菌PCP-1中鉴定出几乎相同的mor基因，此外还有一种可诱导的细胞色素P450，这表明存在水平基因转移。在分枝杆菌属菌株HE5中未发现环状或线性质粒的证据。对morC下游序列的分析表明，分枝杆菌属菌株HE5和分枝杆菌属菌株RP1之间以及嗜氯分枝杆菌之间该基因区域存在差异，这表明重组后发生了插入或缺失。在mor基因的下游，在所有研究菌株中都鉴定出了编码假定谷氨酰胺合成酶的orf1'基因。分枝杆菌属菌株HE5的morC基因进行了异源表达。纯化的重组蛋白FdR(mor)被表征为一种单体44 kDa蛋白，是一种严格依赖NADH、含FAD的还原酶。测定了FdR(mor)对底物NADH（37.7±4.1 microM）以及人工电子受体铁氰化钾（14.2±1.1 microM）和细胞色素c（28.0±3.6 microM）的K(m)值。结果表明，FdR(mor)能与其天然氧化还原伙伴Fe(3)S(4)蛋白Fd(mor)以及Fe(2)S(2)蛋白肾上腺皮质铁氧化还原蛋白在功能上相互作用，尽管效率低得多，但与菠菜铁氧化还原蛋白无相互作用。相反，肾上腺皮质铁氧化还原蛋白的天然氧化还原伙伴肾上腺皮质铁氧化还原蛋白还原酶在活性测定中不能使用Fd(mor)。这些结果表明，FdR(mor)可以利用不同的铁氧化还原蛋白，但Fd(mor)需要来自P450(mor)系统的特异性NADH:铁氧化还原蛋白氧化还原酶FdR(mor)才能实现高效催化功能。
