Ju Yingchen, Tong Shuilong, Gao Yongxiang, Zhao Wei, Liu Qi, Gu Qiong, Xu Jun, Niu Liwen, Teng Maikun, Zhou Huihao
Research Center for Structural Biology and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027, China; Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027, China.
J Struct Biol. 2016 Sep;195(3):306-315. doi: 10.1016/j.jsb.2016.07.008. Epub 2016 Jul 13.
l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications.
L-氨基酸氧化酶/脱氨酶（LAAOs/LAADs）是一类氧化还原酶，可催化L-氨基酸氧化脱氨生成α-酮酸。它们广泛分布于真核生物和原核生物中，具有多样的底物特异性、翻译后修饰和细胞定位。虽然从蛇毒中分离出的LAAOs已得到广泛表征，但其他物种的LAAOs的结构和功能在很大程度上仍不清楚。在此，我们报道了普通变形杆菌（pvLAAD）的一种细菌膜结合LAAD与黄素腺嘌呤二核苷酸（FAD）复合物的晶体结构。我们发现pvLAAD的整体折叠与典型的LAAOs不同。相反，它类似于d-氨基酸氧化酶（DAAOs），在蛋白质表面有一个额外的疏水插入模块。结构分析和脂质体结合试验表明，该疏水模块作为LAADs的一个额外膜结合位点。发现变形杆菌属和普罗威登斯菌属的细菌编码两类膜结合LAADs。基于我们的结构，提出并通过生物化学分析了残基Q278和L317在底物选择性中的关键作用。虽然有人提出膜上的LAADs将电子转移到呼吸链以实现FAD的再氧化，但我们观察到纯化的pvLAAD在体外可产生大量过氧化氢，这表明它可以像典型的LAAOs通常那样利用氧气直接将FADH2再氧化。这些发现为更好地理解这类酶提供了新的见解，并将有助于开发用于工业应用的生物催化剂。
