Mechanistic studies on bioremediation metalloenzymes

 描述(申请人提供)：芳香化合物通常是在化石燃料、溶剂、杀虫剂和塑料的不完全燃烧过程中形成的。它们在生物圈中的位置，无论是有意还是无意，一直是工业化国家的一个问题。 生物修复是指有意识地利用和操纵生物体来去除环境污染物。快速的生长速度、全球丰度和高突变率相结合，使细菌能够适应利用污染物作为碳源。在好氧微生物中，芳香族底物的活化通常通过环的羟基化和随后的脱芳构化来实现。催化这些反应的环分裂双加氧酶含有Fe3+离子(内醇双加氧酶)或Fe2+离子(外醇裂解酶)。最近发现了第三类双加氧酶。这些酶属于Cupin超家族，其特征是具有六链桶折叠和保守的氨基酸基序，为金属离子提供3His或2-或3His-1Glu配体环境。龙胆酸1，2-双加氧酶(GDO)和水杨酸1，2-双加氧酶(SDO)属于这类新的酶，它们含有一个3-His金属结合部位。已经报道了一些GDO的机械学工作，然而，基本上还没有发表SDO的机械学工作。这为利用模型研究SDO的机制提供了机会，因为由于底物的结构差异，SDO不太可能与GDO相同。此外，还发现了一种氧化2，4‘-二羟基苯乙酮的生物修复酶(2，4’-二羟基苯乙酮双加氧酶，DAD)。这种丘比素酶含有3-His-1-Glu活性部位。合成模型研究也有助于阐明这种新酶的机制。在这项提议中，我们的目标是使用不同性质的有机配体来合成铁化合物。我们将合成三个含氮和三个含氮一羧酸的配体。这些配体将与铁(II)和铁(III)反应生成具有可调配位几何结构和电子性质的模型体系。将对这些配合物进行系统的研究，以建立模型络合物结构与光谱和物理性质之间的关联。被认为是选定生物修复酶的良好结构和光谱模型的化合物将被研究仿生活性。接下来，我们将探讨模型络合物对含芳环化合物的反应活性。将进行测试，以测试络合物的化学计量和催化双加氧酶活性。如果在低温下观察到中间体，我们将尝试分离和鉴定这些物种。这些信息将提供与生物修复酶有关的机械性见解，从而更好地了解这些新类别的酶如何发挥其功能。

 DESCRIPTION (provided by applicant): Aromatic compounds are commonly formed during the incomplete burning of fossil fuels, solvents, pesticides and plastics. Their placement within the biosphere either intentionally or accidentally has been a problem for industrialized countries. Bioremediation is the intentional use and manipulation of living organisms to remove environ- mental pollutants. The combination of rapid growth rate, global abundance and high rate of mutations enables bacteria to adapt to utilize pollutants as a carbon source. In aerobic microorganisms, activation of an aromatic substrate is usually effected by hydroxylation of the ring and subsequent dearomatization. Ring-fission dioxygenases that catalyze these reactions contain Fe3+ ions (intradiol dioxygenases) or Fe2+ ions (extradiol cleaving enzymes). A third Class of dioxygenases has been recently identified. These enzymes belong to the cupin super- family, which is characterized by a six-stranded -barrel fold and conserved amino acid motifs providing 3His or 2- or 3His-1Glu ligand environments to metal ions. The enzymes gentisate 1,2-dioxygenase (GDO) and salicylate 1,2-dioxygenase (SDO) belong to this new class and contain a 3-His metal binding site. Some mechanistic work has been reported for GDO, however, essentially no mechanistic work for SDO has been published. This provides an opportunity to investigate the mechanism for SDO using model studies since it is unlikely to be the same as GDO due to structural differences in the substrates. Additionally, a scarcely characterize bioremediation enzyme (2,4'-dihydroxyacetophenone dioxygenase, DAD) capable of oxidizing 2,4'-dihydroxyacetophenone (DHAP) has been identified. This cupin enzyme contains a 3-His-1-Glu active site. Synthetic model studies can assist in elucidating the mechanism for this new enzyme as well. In this proposal we aim to synthesize iron compounds using organic ligands of varying properties. We will synthesize three nitrogen and three nitrogen-one-carboxylate ligands. These ligands will be reacted with iron(II) and iron(III) to generate model systems with tunable coordination geometry and electronic properties. The complexes will be systematically studied to generate correlations be- tween the model complex structure and spectroscopic and physical properties. Compounds which are deemed good structural and spectroscopic models for selected bioremediation enzymes will be studied for biomimetic activity. Next, we will probe the reactivity of model complexes towards aromatic ring-containing compounds. Assays will be performed to test the complexes for both stoichiometric and catalytic dioxygenase activity. If intermediates are observed at low temperature, we will attempt to isolate and characterize these species. Such information will provide mechanistic insights relevant to bioremediation enzymes thus providing a better understanding of how these new classes of enzymes performs their function.