Carboxylation of phenolic compounds

酚类化合物是最常见的天然化合物之一，因此是微生物的重要底物。它们的有氧代谢涉及加氧酶和作为共底物的氧。在没有氧气的情况下，需要完全不同的反应和酶。在模式细菌Thauera aromatica和Azoarcus sp. EbN1中，其基因组已被测序，苯酚首先被磷酸化，然后被羧化，产生4-羟基苯甲酸酯。这项建议有两个目的。(1)本文将对厌氧苯酚代谢的新酶--苯磷酸合成酶和苯磷酸羧化酶进行详细的研究。在这两种情况下，预期都有酶结合的中间体，即分别为酶-磷酸盐和酶-酚盐。苯磷酸羧化酶是一个新的羧化酶/脱羧酶家族的成员，其作用于酚类化合物，使用CO2作为底物，不含生物素或二磷酸硫胺素，需要K+和二价金属阳离子（Mg 2+或Mn 2+）才能活性，并且被氧气迅速灭活。(2)编码这两种酶的七个基因位于一个大的苯酚诱导的操纵子上，该操纵子含有八个功能未知的额外基因。随后，将检测它们是否参与相关酚类化合物的代谢。本研究将探讨不同酚类化合物如邻苯二酚或邻甲酚的羧化反应，以及酚酸如原儿茶酸、香草酸和龙胆酸的进一步代谢。这些结果有望为新的反应和酶提供见解，有助于了解酚类化合物在缺氧栖息地特别是地下水和肠道中的转化，并导致羧化系统的生物技术应用。

Phenolic compounds represent one of the most common natural compounds and therefore are important substrates for microorganisms. Their aerobic metabolism involves oxygenases and oxygen as a cosubstrate. In the absence of oxygen completely different reactions and enzymes are required. In the model bacteria Thauera aromatica and Azoarcus sp. EbN1, whose genome has been sequenced, phenol is first phosphorylated and then carboxylated yielding 4- hydroxybenzoate. This proposal has two aims. (1) The new enzymes of anaerobic phenol metabolism, phenylphosphate synthase and phenylphosphate carboxylase, will be studied in detail. In both cases enzyme-bound intermediates are to be expected, i.e. enzyme-phosphate and enzyme-phenolate, respectively. Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are rapidly inactivated by oxygen. (2) The seven genes coding for these two enzymes are located on a large phenol induced operon which contains eight additional genes of unknown function. Lateron, it will be testet whether they are involved in the metabolism of related phenolic compounds. The carboxylation of various phenolic compounds such as catechol or ortho-cresol and the further metabolism of phenolic acids such as protocatechuate, vanillate and gentisate will be investigated. The results promise to provide insights into novel reactions and enzymes, to contribute to the understanding of the transformations of phenolic compounds in anoxic habitats notably in ground water and intestinal tract, and to lead to the biotechnological application of the carboxylation systems.