Possible radical mechanism in enzymatic carboxylation of phenol

苯酚的厌氧代谢是通过对羧化生成4-羟基苯甲酸酯进行的。这种增能反应在化学上很早就知道了(Kolbe-Schmitt-羧化)，但在生物学上研究得很少。将研究芳香梭菌中的苯酚羧化酶系统。它不属于所研究的任何一类羧基酶，因为它通过磷酸化的自由中间体(苯基磷酸)分两步进行，并使用金属(Mn2+)作为助催化剂，二氧化碳作为底物。虽然苯酚的磷酸化似乎是推动羧化的关键，但它使苯基磷酸不是二氧化碳攻击的不良底物。苯酚的羧化体系不依赖于生物素或硫胺素二磷酸，并且是复杂的；10种蛋白质是由苯酚诱导的。羧基酶属于一个蛋白质家族，其功能在很大程度上是未知的。最值得注意的是，该酶对氧和自由基捕捉剂极其敏感。苯基磷酸酯羧化酶将被提纯和鉴定。我们将特别关注催化机理，根据我们的工作假设，催化机理涉及一种强烈结合酶的酚酸盐中间体和一种自由基物种。

Anaerobic metabolism of phenol proceeds via para carboxylation to 4-hydroxybenzoate. This endergonic reaction is known since long in chemistry (Kolbe-Schmitt-carboxylation), but is only poorly studied in biology. The phenol carboxylating enzyme system in the bacterium Thauera aromatica will be investigated. It does not belong to any group of the studied carboxylases as it proceeds in two steps via a phosphorylated free intermediate (phenylphosphate) and uses a metal (Mn2+) as co-catalyst and carbon dioxide as substrate. While phosphorylation of phenol seems essential to drive carboxylation forward, it makes phenylphosphate a poor substrate for CO2 attack. The phenol carboxylating system does not depend on biotin or thiamine diphosphate and is complex; 10 proteins are phenol-induced. The carboxylase belongs to a family of proteins whose functions are largely unknown. Most remarkably, the enzyme is extremely sensitive to oxygen and radical trapping agents. The phenyl-phosphate carboxylating enzyme will be purified and characterized. Special focus will be on the catalytic mechanism which according to our working hypothesis involves a strongly enzyme-bound phenolate intermediate and a radical species.