Molecular analysis of the soluble butane monooxygenase from 'Pseudomonas butanovora'.
Molecular analysis of the soluble butane monooxygenase from 'Pseudomonas butanovora'.
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DOI：
10.1099/00221287-148-11-3617
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发表时间：
2002-11
期刊：
影响因子：
1.5
通讯作者：
M. Sluis;L. Sayavedra-Soto;D. Arp
中科院分区：
文献类型：
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作者：
M. Sluis;L. Sayavedra-Soto;D. Arp
“噬丁烷假单胞菌”能够通过丁烷氧化成1-丁醇与丁烷一起生长，这是由可溶性丁烷单加氧酶（sBMO）催化的。乙烯（sBMO的替代底物）的体外氧化在细胞提取物的可溶性部分中重建，并且是NADH依赖性的。丁烷单加氧酶被分离为三个组分，它们是底物氧化所必需的。与丁烷单加氧酶相关的肽的N-末端序列导致5797个核苷酸的bmo基因簇的克隆和测序。与其他多组分单加氧酶的推导的氨基酸序列的比较表明，sBMO是一个多聚体羟化酶与61，45和19 kDa的亚基编码的bmoXYZ，40 kDa的氧化还原酶编码的bmoC，和15 kDa的调节蛋白编码的bmoB。第六个结构基因（bmoD）编码一个9.6 kDa的蛋白质与mmoD（orfY），一个假定的金属中心组装蛋白的可溶性甲烷单加氧酶的相似性。bmoX的插入失活导致不能用丁烷生长的突变体“P. butanovora”菌株。与sigma（54）依赖性转录起始相关的启动子的特征性启动子元件位于bmo基因的上游。在丁烷诱导的细胞中检测到所有六个基因的表达。丁烷单加氧酶从'P. butanovora'对齐最密切的非血红素羧酸桥接的二铁单加氧酶，而且，包含特征性的铁结合基序。丁烷单加氧酶和甲烷单加氧酶的肽之间的高序列同一性（高达64%）的结构和机制的影响进行了讨论。
'Pseudomonas butanovora' is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO). In vitro oxidation of ethylene (an alternative substrate for sBMO) was reconstituted in the soluble portion of cell extracts and was NADH-dependent. Butane monooxygenase was separated into three components which were obligately required for substrate oxidation. The N-terminal sequences of the peptides associated with butane monooxygenase led to the cloning and sequencing of the 5797 nucleotide bmo gene cluster. Comparisons of the deduced amino acid sequences with other multicomponent monooxygenases suggest that sBMO is a multimeric hydroxylase with 61, 45 and 19 kDa subunits encoded by bmoXYZ, a 40 kDa oxidoreductase encoded by bmoC, and a 15 kDa regulatory protein encoded by bmoB. A sixth structural gene (bmoD) encodes a 9.6 kDa protein with similarity exclusively to mmoD (orfY), a putative metal centre assembly protein of the soluble methane monooxygenases. Insertional inactivation of bmoX resulted in a mutant 'P. butanovora' strain incapable of growth with butane. A putative promoter element characteristic of promoters associated with sigma(54)-dependent transcription initiation was located upstream of the bmo genes. Expression of all six genes was detected in butane-induced cells. Butane monooxygenase from 'P. butanovora' aligns most closely with non-haem carboxylate-bridged diiron monooxygenases and, moreover, contains the characteristic iron-binding motif. The structural and mechanistic implications of the high sequence identity (up to 64%) between the peptides of butane monooxygenase and methane monooxygenases are discussed.