Tungsten-dependent aldehyde oxidoreductases: specific W-cofactor insertion and interactions with Fe-S clusters in catalysis and maturation

醛：铁氧还蛋白氧化还原酶（AOR）属于一个含钨酶家族，主要来自超嗜热古菌。它们含有一个钨-铋-钼辉素辅助因子作为活性位点的一部分，在遗传和结构上与其他三个含钼辉素酶家族不同，后者主要含有钼而不是钨。我们最近在中温反硝化细菌Aromatoleum aromaticum中发现了一种不寻常的氧稳定AOR同源物，它将各种芳香或脂肪醛氧化成相应的酸。纯化后的蛋白由一个含有w -辅因子和一个Fe4S4簇的大α亚基、一个含有四个Fe4S4簇的小β亚基和一个含有FAD的中γ亚基组成。这种复杂的组成和对氧的不敏感明显不同于已知的古细菌同源物的性质，证明了对酶的更彻底的表征。我们打算表征这种新型AOR同工酶的催化和结构性质，特别是关于由各种Fe-S簇形成的表观电子传递继电器的功能。这些研究的重点将放在反应的可逆性和氧稳定性增加的原因上。此外，将建立一个重组表达系统来获得活性AOR和突变变体，这将允许评估单个Fe-S簇或其他活性位点残基对催化机制的贡献。该项目的第二个主要方面将是w -辅因子的生物合成和纳入AOR的机制。a . aromaticum与AOR同时合成含有功能mo -辅因子的酶，需要一种特定的合成机制，并将两种辅因子结合到正确的酶中。为了进一步阐明这一过程，我们将研究基因组中编码为复制版本的辅助因子合成酶，特别是插入钼/钨的MoeA蛋白和引入钼酸盐辅助因子SH基团所需的MoaD蛋白。我们将研究纯化的蛋白质，我们将尝试将其重组成体外W-辅因子生物合成系统，并将产生缺乏相应基因的突变体来研究其体内效应。特别是，我们将研究一种假设的w -辅因子成熟的新途径，该途径涉及AOR及其Fe-S簇作为支架系统。

Aldehyde:ferredoxin oxidoreductases (AOR) belong to a family of tungsten-containing enzymes mostly known from hyperthermophilic Archaea. They contain a tungsten-bis-molybdopterin cofactor as part of the active site and are genetically and structurally distinct from the other three enzyme families of molybdopterin-containing enzymes, which mostly contain molybdenum instead of tungsten. We have recently discovered an unusually oxygen-stable AOR ortholog in the mesophilic denitrifying bacterium Aromatoleum aromaticum, which oxidises a variety of aromatic or aliphatic aldehydes to the corresponding acids. The purified protein consists of a large alpha subunit containing the W-cofactor and one Fe4S4 cluster, a small beta subunit containing four Fe4S4 clusters and a medium gamma subunit containing FAD. This complex composition and the insensitivity against oxygen differs markedly from the properties of the known archaeal orthologs and justifies a more thorough characterization of the enzyme. We intend to characterize the catalytic and structural properties of this new type of AOR isoenzyme, especially regarding the function of the apparent electron transfer relay formed by the various Fe-S clusters. The focus of these studies will be on the reversibility of the reaction and the reason for the increased oxygen stability. In addition, a recombinant expression system will be established to obtain active AOR and mutant variants, which will allow to evaluate the contributions of individual Fe-S clusters or other active site residues to the catalytic mechanism. A second main aspect of the project will be the mechanism of W-cofactor biosynthesis and incorporation into AOR. A. aromaticum synthesizes functional Mo-cofactor containing enzymes at the same time as AOR, necessitating a specific machinery of synthesis and incorporation of both cofactors into the correct enzymes. To shed more light on this process, we will study the enzymes of cofactor synthesis that are encoded in duplicated versions in the genome, particularly the Mo-/W-inserting MoeA proteins and the MoaD proteins required for introducing the SH groups of the molybdopterin cofactors. We will study the purified proteins which we will try to reconstitute into a system of in-vitro W- cofactor biosynthesis, and will also generate mutants lacking the respective genes to study their in-vivo effects. Particularly, we will investigate a hypothetical new pathway of W-cofactor maturation that involves AOR and its Fe-S clusters as scaffold system.