Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase
Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase
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DOI：
10.1128/jb.184.6.1547-1555.2002
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发表时间：
2002-03
影响因子：
3.2
通讯作者：
N. Zhou;J. Al-Dulayymi;M. Baird;Peter A. Williams
中科院分区：
文献类型：
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作者：
N. Zhou;J. Al-Dulayymi;M. Baird;Peter A. Williams
对萘降解菌Ralstonia sp. U2的加氧酶簇nagAaGHAbAcAd基因进行了克隆和高效表达。水杨酸5-羟化酶（S5 H）的活性，转化水杨酸龙胆酸盐，是目前在体外仅在单一提取物的细胞与过表达的nagAaGHAb或在混合物中的三个细胞提取物分别含有，NagGH（加氧酶组分），NagAa（铁氧还蛋白还原酶），和NagAb（铁氧还蛋白）。S5 H活性所需的三种提取物中的每一种在过量的其他提取物存在下都是速率限制的，但是当过量时，不影响催化速率。S5 H催化3-和4-取代水杨酸酯芳环的5-羟基化反应。然而，5-甲基水杨酸酯的甲基被羟基化以产生5-羟甲基衍生物，并且5-氯水杨酸酯环上的6位被羟基化，产生5-氯-2，6-二羟基苯甲酸酯。在基于NADH的吲哚连接氧化的nag萘双加氧酶（NDO）的测定中，三种提取物对于活性是必需的（NagAcAd、NagAa和NagAb）。NDO和S5 H在来自“经典”萘降解菌P. putida NCIMB 9816的nag蛋白和相应nah NDO蛋白的所有可能组合存在下测定。所有三种加氧酶组分与来自任一菌株的电子传递蛋白的混合组合起作用。来自菌株U2的S5 H是一种独特的单加氧酶，其与双加氧酶如NDO具有序列相似性，但在结构上也足够相似以与相同的电子传递链相互作用，并且可能在菌株U2中的萘催化过程中在体内这样做。
ABSTRACT The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the “classical” naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.