Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.
Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.
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DOI：
10.1371/journal.pone.0127288
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Dey M
中科院分区：
文献类型：
--
作者：
Brummett AE;Schnicker NJ;Crider A;Todd JD;Dey M
渗透剂二甲基磺基丙酸盐（DMSP）是海洋环境中的关键营养素，其通过被称为DMSP裂解酶的酶被细菌催化产生二甲基硫（DMS），二甲基硫是一种在气候调节、硫循环和向高等生物发出信号中重要的气体。尽管DMSP裂解酶的环境意义，很少有人知道他们如何在机制水平上发挥作用。在这项研究中，我们的生物化学特性DddW，从模式玫瑰Ruegeria pomeroyi DSS-3的DMSP裂解酶。DddW是一种16.9 kDa的酶，含有C-末端cupin结构域，并从DMSP底物中释放丙烯酸酯、质子和DMS。我们的研究表明，作为纯化的DddW是一种金属酶，像DddQ和DddP DMSP裂解酶，但含有铁辅因子。金属辅因子是DddW DMSP裂解酶活性所必需的，因为添加金属螯合剂EDTA废除了其酶活性，cupin基序中的关键金属结合残基（His 81，His 83，Glu 87和His 121）的取代突变也是如此。金属结合亲和力和催化活性的测量表明，Fe（II）最有可能是具有纳摩尔结合亲和力的优选催化金属离子。化学计量研究表明，DddW需要一个Fe（II）每个单体。电子吸收和电子顺磁共振（EPR）的研究表明，NO和Fe（II）-DddW之间的相互作用，与NO结合到EPR沉默的Fe（II）网站产生EPR活性物种（g = 4.29，3.95，2.00）。的EPR信号的菱形的变化中观察到的DMSP的存在下，表明基板结合到铁网站没有取代绑定NO。这项工作提供了深入了解DMSP裂解DddW催化的机制。
The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.
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