Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043
Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043
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DOI：
10.1128/aem.00377-18
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发表时间：
2018-04
影响因子：
4.4
通讯作者：
Yahui Shao;Lizhong Guo;Yu-qing Zhang;Hao Yu;Baisuo Zhao;Hai-Qiang Pang;W. Lu
中科院分区：
文献类型：
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作者：
Yahui Shao;Lizhong Guo;Yu-qing Zhang;Hao Yu;Baisuo Zhao;Hai-Qiang Pang;W. Lu
摘要：虽然一些细菌，包括 Chromohalobacter salexigens DSM 3043，可以使用甘氨酸甜菜碱 (GB) 作为碳和能量的唯一来源，但有关 GB 降解途径初始步骤中涉及的基因及其编码蛋白的信息很少。在本研究中，保守域分析、框内缺失突变体的构建和体内功能互补测定的结果表明，开放阅读框Csal_1004和Csal_1005（分别称为bmoA和bmoB）可能充当新型Rieske型加氧酶系统中的末端加氧酶和铁氧还蛋白还原酶基因，将C. salexigens DSM中的GB转化为二甲基甘氨酸3043. 为了进一步验证其功能，BmoA和BmoB在大肠杆菌中异源过表达，13C核磁共振分析表明二甲基甘氨酸在表达BmoAB或BmoA的大肠杆菌BL21(DE3)中积累。此外，His 标记的 BmoA 和 BmoB 分别纯化至电泳均质，并分别估计为同源三聚体和单体。体外生化分析表明，BmoB 是一种 NADH 依赖性黄素还原酶，以一个非共价结合的黄素腺嘌呤二核苷酸（FAD）作为其辅基。在 BmoB、NADH 和黄素存在下，BmoA 可以有氧地将 GB 降解为二甲基甘氨酸，同时产生甲醛。 BmoA对GB表现出严格的底物特异性，其去甲基化活性受到Fe2+的刺激。系统发育分析表明，BmoA属于Rieske非血红素铁加氧酶(RO)家族的V族，该族的所有成员都能够使用季铵化合物作为底物。重要性 GB 在自然界中分布广泛。除了作为相容性溶质在细胞内积累以应对渗透压之外，它还可以被许多细菌用作碳和能量的来源。然而，目前关于细菌需氧 GB 降解途径初始步骤的分子和生化机制的知识非常有限。在这里，我们报告了一种新型双组分 Rieske 型单加氧酶系统 GB 单加氧酶 (BMO) 的分子和生化特征，该系统负责 C. salexigens DSM 3043 中 GB 氧化去甲基化为二甲基甘氨酸。这项研究获得的结果扩展了我们对微生物 GB 降解为二甲基甘氨酸的催化反应的认识。
ABSTRACT Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+. Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates. IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.