Oxidative Maturation and Structural Characterization of Prenylated FMN Binding by UbiD, a Decarboxylase Involved in Bacterial Ubiquinone Biosynthesis
Oxidative Maturation and Structural Characterization of Prenylated FMN Binding by UbiD, a Decarboxylase Involved in Bacterial Ubiquinone Biosynthesis
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DOI：
10.1074/jbc.m116.762732
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发表时间：
2017-03-10
影响因子：
4.8
通讯作者：
Leys, David
中科院分区：
文献类型：
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作者：
Marshall, Stephen A.;Fisher, Karl;Leys, David
可逆脱羧酶Fdc 1的活性依赖于异戊二烯化FMN（prFMN），这是一种最近发现的辅因子。氧化的prFMN支持1，3-偶极环加成机制，该机制支持可逆脱羧。Fdc 1是UbiD酶家族的一个独特成员，典型的UbiD催化对羟基苯甲酸型底物的羧化（脱羧）。在这里，我们表明，大肠杆菌UbiD酶，这是在泛醌生物合成的牵连，不能在一个活跃的全酶的形式分离，尽管事实上活跃holoFdc 1很容易获得。holoUbiD的形成需要在体外用还原的prFMN重建apoUbiD。此外，虽然Fdc 1脱辅基酶可以很容易地重建和激活，在体外氧化成成熟的prFMN辅因子失速在形成自由基prFMN物种在holoUbiD。进一步的体外氧化成熟仅在碱性pH下发生，表明质子偶联电子转移先于完全氧化的prFMN的形成。holoUbiD的晶体结构揭示了一个相对开放的活性位点，可能通过域运动从溶剂中被封闭。在UbiD晶体结构之一中存在prFMN亚硫酸盐加合物证实了氧化成熟确实在环境pH下以缓慢的时间尺度发生。在一系列假定的对羟基苯甲酸底物中，未检测到活性。然而，所提出的泛醌前体底物的八异戊二烯尾部缺乏明显的疏水结合口袋确实表明UbiD可能作用于非异戊二烯化的前体。我们的数据揭示了一个意想不到的变化发生在域的流动性，prFMN结合，并通过UbiD酶家族的成熟。
The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de) carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holoenzyme form despite the fact active holoFdc1 is readily obtained. Formation of holoUbiD requires reconstitution in vitro of the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturation in vitro occurs only at alkaline pH, suggesting a proton-coupled electron transfer precedes formation of the fully oxidized prFMN. Crystal structures of holoUbiD reveal a relatively open active site potentially occluded from solvent through domain motion. The presence of a prFMN sulfite-adduct in one of the UbiD crystal structures confirms oxidative maturation does occur at ambient pH on a slow time scale. Activity could not be detected for a range of putative para-hydroxybenzoic acid substrates tested. However, the lack of an obvious hydrophobic binding pocket for the octaprenyl tail of the proposed ubiquinone precursor substrate does suggest UbiD might act on a non-prenylated precursor. Our data reveals an unexpected variation occurs in domain mobility, prFMN binding, and maturation by the UbiD enzyme family.