Deller Sigrid, Sollner Sonja, Trenker-El-Toukhy Rosemarie, Jelesarov Ilian, Gübitz Georg M, Macheroux Peter
Institute of Biochemistry, Graz University of Technology, Petersgasse 12/II, A-8010 Graz, Austria.
Biochemistry. 2006 Jun 13;45(23):7083-91. doi: 10.1021/bi052478r.
The gene yhdA from Bacillus subtilis encoding a putative flavin mononucleotide (FMN)-dependent oxidoreductase was cloned and heterologously expressed in Escherichia coli. The purified enzyme has a noncovalently bound FMN cofactor, which is preferentially reduced by NADPH, indicating that YhdA is a NADPH:FMN oxidoreductase. The rate of NADPH oxidation is enhanced by the addition of external FMN, and analysis of initial rate measurements reveals the occurrence of a ternary complex in a bi-bi reaction mechanism. YhdA has also been shown to reductively cleave the -N=N- bond in azo dyes at the expense of NADPH, and hence, it possesses azoreductase activity, however, at a rate 100 times slower than that found for FMN. Using Cibacron Marine as a model compound, we could demonstrate that the dye is a competitive inhibitor of NADPH and FMN. The utilization of NADPH and the absence of a flavin semiquinone radical distinguish YhdA from flavodoxins, which adopt the same structural fold, i.e., a five-stranded beta sheet sandwiched by five alpha helices. The native molecular-mass of YhdA was determined to be 76 kDa, suggesting that the protein occurs as a tetramer, whereas the YhdA homologue in Saccharomyces cerevisiae (YLR011wp) forms a dimer in solution. Interestingly, the different oligomerization of these homologous proteins correlates to their thermostability, with YhdA exhibiting a melting point of 86.5 degrees C, which is 26.3 degrees C higher than that for the yeast protein. This unusually high melting point is proposed to be the result of increased hydrophobic packing between dimers and the additional presence of four salt bridges stabilizing the dimer-dimer interface.
克隆了来自枯草芽孢杆菌的编码假定黄素单核苷酸（FMN）依赖性氧化还原酶的基因yhdA，并在大肠杆菌中进行了异源表达。纯化后的酶具有非共价结合的FMN辅因子，该辅因子优先被NADPH还原，这表明YhdA是一种NADPH：FMN氧化还原酶。添加外部FMN可提高NADPH的氧化速率，对初始速率测量结果的分析揭示了双底物反应机制中三元复合物的存在。YhdA还被证明能以NADPH为代价还原偶氮染料中的-N=N-键，因此它具有偶氮还原酶活性，不过其反应速率比FMN慢100倍。以汽巴克隆海洋蓝为模型化合物，我们证明该染料是NADPH和FMN的竞争性抑制剂。YhdA对NADPH的利用以及不存在黄素半醌自由基，使其与黄素氧还蛋白不同，黄素氧还蛋白具有相同的结构折叠，即由五个α螺旋夹着的五股β折叠。YhdA的天然分子量测定为76 kDa，表明该蛋白以四聚体形式存在，而酿酒酵母中的YhdA同源物（YLR011wp）在溶液中形成二聚体。有趣的是，这些同源蛋白不同的寡聚化状态与其热稳定性相关，YhdA的熔点为86.5℃，比酵母蛋白高26.3℃。这种异常高的熔点被认为是二聚体之间疏水堆积增加以及稳定二聚体-二聚体界面的四个盐桥额外存在的结果。
