Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica
Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica
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DOI：
10.1128/jb.183.3.968-979.2001
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发表时间：
2001-02-01
影响因子：
3.2
通讯作者：
Fuchs, G
中科院分区：
文献类型：
--
作者：
Laempe, D;Jahn, M;Fuchs, G
研究了3-羟基苯甲酸酯在芳香奇异果（Thauera aromatica）发酵菌中的厌氧代谢。用这种基质生长的细胞适应于用苯甲酸盐生长，但不适应于用4-羟基苯甲酸盐生长。反之亦然，4-羟基苯甲酸生长的细胞不利用3-羟基苯甲酸。3-羟基苯甲酸代谢的第一步是辅酶A（CoA）硫酯形成，其由诱导型3-羟基苯甲酸-CoA连接酶催化。对该酶进行了纯化和表征。细胞提取物进一步代谢3-羟基苯甲酰基-CoA需要MgATP，并与每摩尔添加的底物氧化2摩尔还原紫精染料偶联。3-羟基苯甲酰基-CoA还原酶的纯化揭示了这种活性是由于苯甲酰基-CoA还原酶，其几乎与苯甲酰基-CoA一样有效地还原3-羟基类似物。含有羟基取代的脂环族二烯酰辅酶A产物的进一步代谢显然需要额外的特异性酶。3-羟基苯甲酸盐生长的细胞与苯甲酸盐生长的细胞的蛋白质模式的比较，揭示了几个3-羟基苯甲酸盐诱导的蛋白质，K-末端氨基酸序列的诱导蛋白质进行了测定，并确定相应的基因进行测序。一个由6个相邻基因组成的簇包含底物诱导蛋白1至3的基因;这个簇可能还不完整。蛋白质1是一种短链醇脱氢酶。蛋白质2是烯酰辅酶A水合酶的成员。蛋白3被鉴定为3-羟基苯甲酸-CoA连接酶。蛋白4是烯酰辅酶A水合酶的另一个成员。此外，存在三个编码β-氧化酶的基因。这里的厌氧3-羟基苯甲酸酯代谢显然将一般苯甲酰-CoA途径的酶（苯甲酰-CoA还原酶）和电子载体（铁氧还蛋白）与对3-羟基苯甲酸酯途径特异的酶结合。这就提出了一些关于这两种途径调节的问题。
The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown a cells did not utilize 3-hydroxybenzoate. The first step in 3-hydrorcybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the K-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta -oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.