A Bacterial Multidomain NAD-Independent D-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for D-Lactate Oxidization
A Bacterial Multidomain NAD-Independent D-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for D-Lactate Oxidization
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细菌多域 NAD 依赖性 D-乳酸脱氢酶利用黄素腺嘌呤二核苷酸和 Fe-S 簇作为辅因子，并利用醌作为 D-乳酸氧化的电子受体
DOI：
10.1128/jb.00342-17
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发表时间：
2017-11-01
影响因子：
3.2
通讯作者：
Gao, Chao
中科院分区：
文献类型：
--
作者：
Jiang, Tianyi;Guo, Xiaoting;Gao, Chao
细菌膜相关NAD非依赖性d-乳酸脱氢酶（Fe-S d-iLDH）可将d-乳酸氧化为丙酮酸。该酶的序列分析表明，它含有一个Fe-S氧化还原酶结构域，除了黄素腺嘌呤二核苷酸（FAD）含有脱氢酶结构域，这不同于其他典型的d-iLDH。来自恶臭假单胞菌KT 2440的Fe-S d-iLDH被纯化为带有His标签的蛋白质并进行了详细表征。这种单体酶表现出与l-乳酸和几种d-2-羟基酸的活性。醌被证明是该酶的首选电子受体。然后将酶的两个结构域分别异源表达和纯化。通过对Fe-S氧化还原酶结构域的定点突变，初步验证了序列比对预测的Fe-S簇结合基序。含有FAD的脱氢酶结构域保留2-羟基酸氧化活性，尽管与完整的Fe-S d-iLDH相比其降低。与完整的酶相比，含FAD的脱氢酶结构域显示出以细胞色素c为电子受体的催化效率增加，但它完全失去了使用辅酶Q10的能力。此外，含FAD的脱氢酶结构域不再与细胞膜结合，并且它不能支持利用d-乳酸作为碳源。基于所获得的结果，我们得出结论，Fe-S氧化还原酶结构域的功能作为电子传递组件，以促进利用醌作为电子受体的Fe-S d-iLDH，它有助于酶与细胞膜。这些功能使得Fe-S氧化还原酶结构域对于Fe-S d-iLDH的体内乳酸利用功能至关重要。重要性乳酸代谢在生活的大多数领域中起着多方面的作用。乳酸盐利用过程依赖于某些酶将乳酸盐氧化成丙酮酸盐。近年来，新型细菌乳酸氧化酶不断被报道，其中包括独特的非NAD依赖性d-乳酸脱氢酶，该酶除了典型的含黄素结构域（Fe-S d-iLDH）外，还含有Fe-S氧化还原酶结构域。虽然Fe-S d-iLDH在细菌中广泛分布，但对其研究还不够深入。恶臭假单胞菌KT 2440的Fe-S d-iLDH可能是这类酶的代表，它是该菌株的主要d-乳酸氧化酶。对它的研究将有助于了解乳酸利用过程的详细机制。
ABSTRACT Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10. Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivod-lactate utilization function of Fe-S d-iLDH. IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.