Rudolphi A, Tschech A, Fuchs G
Abteilung Angewandte Mikrobiologie, Universität Ulm, Federal Republic of Germany.
Arch Microbiol. 1991;155(3):238-48. doi: 10.1007/BF00252207.
The initial reactions in anaerobic metabolism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o- or p-cresol as sole organic substrate with a generation time of 11 h; o- or p-cresol was completely oxidized to CO2 with nitrate being reduced to N2. A denitrifying Pseudomonas-like strain oxidized m- or -p-cresol as the sole organic growth substrate completely to CO2 with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps: (1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol----4-OH-benzaldehyde----4-OH-benzoate----4-OH-benzoyl-CoA----be nzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating). (2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH3-benzoyl-CoA as central intermediate as follows: o-cresol----4-OH-3-CH3-benzoate----4-OH-3-CH3-benzoyl-CoA----3-CH3-benzo yl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between 14CO2 and the carboxyl of 4-OH-3-CH3-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH3-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH3-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH3-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH3-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH3-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH3-benzoate and further to 3-CH3-benzoyl-CoA. (3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4- and 3,4-dimethylphenol were oxidized to 4-OH-3-CH3- and 4-OH-2-CH3-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.
利用反硝化细菌研究了甲基苯酚（甲酚）和二甲基苯酚厌氧代谢的初始反应。新分离出的一株菌，可能是副球菌属，能够以邻甲酚或对甲酚作为唯一有机底物生长，代时为11小时；邻甲酚或对甲酚被完全氧化为二氧化碳，同时硝酸盐被还原为氮气。一株类似反硝化假单胞菌的菌株以间甲酚或对甲酚作为唯一有机生长底物，代时为14小时，能将其完全氧化为二氧化碳。中间产物的鉴定和/或酶活性的体外测定表明有以下酶促步骤：（1）两种菌株都通过苯甲酰辅酶A作为中心中间体代谢对甲酚，具体如下：对甲酚→4-羟基苯甲醛→4-羟基苯甲酸→4-羟基苯甲酰辅酶A→苯甲酰辅酶A。对甲酚甲基羟化酶催化甲基氧化为4-羟基苯甲醛。醛氧化为4-羟基苯甲酸后，4-羟基苯甲酰辅酶A合成酶将其转化为4-羟基苯甲酰辅酶A；随后4-羟基苯甲酰辅酶A还原酶（脱羟基）催化4-羟基苯甲酰辅酶A还原脱羟基生成苯甲酰辅酶A。（2）在类似副球菌的菌株中，邻甲酚通过3-甲基苯甲酰辅酶A作为中心中间体代谢，具体如下：邻甲酚→4-羟基-3-甲基苯甲酸→4-羟基-3-甲基苯甲酰辅酶A→3-甲基苯甲酰辅酶A。已证实有以下几种酶：（a）一种催化14CO2与4-羟基-3-甲基苯甲酸羧基之间同位素交换反应的酶；该活性被认为是邻甲酚羧化酶催化的部分反应。（b）4-羟基-3-甲基苯甲酰辅酶A合成酶（生成AMP）将羧化产物4-羟基-3-甲基苯甲酸激活为其辅酶A硫酯。（c）4-羟基-3-甲基苯甲酰辅酶A还原酶（脱羟基）以还原型苄基紫精作为电子供体，催化4-羟基的还原脱羟基反应，生成3-甲基苯甲酰辅酶A。当3-甲基苯甲酸代谢时，该硫酯也可能由辅酶A连接酶的作用形成。2,4-二甲基苯酚通过4-羟基-3-甲基苯甲酸进一步代谢为3-甲基苯甲酰辅酶A。（3）在类似假单胞菌的菌株中，间甲酚厌氧代谢的初始反应尚未明确。未发现甲基氧化或间甲酚羧化的迹象。相反，2,4-二甲基苯酚和3,4-二甲基苯酚分别被氧化为4-羟基-3-甲基苯甲酸和4-羟基-2-甲基苯甲酸，可能是由对甲酚甲基羟化酶引发的；然而，这些化合物未进一步代谢。
