Bernat B A, Laughlin L T, Armstrong R N
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 1997 Mar 18;36(11):3050-5. doi: 10.1021/bi963172a.
The enzyme conferring resistance to the antibiotic fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] originally reported by Suarez and co-workers [Area, P., Hardisson, C., & Suarez, J. E. (1990) Antimicrob. Agents Chemother. 34, 844-848] is demonstrated in this study to be a metalloglutathione transferase. The apoenzyme is a dimer of 16 kDa subunits. Electron paramagnetic resonance spectroscopy and water proton nuclear magnetic resonance longitudinal relaxation rates suggest that each subunit contains a mononuclear Mn2+ center that interacts strongly with the substrate fosfomycin (Kd = 17 microM) more weakly with the product (Kd = 1.1 mM) and very weakly or not at all with GSH. Inhomogeneous broadening of the EPR signals of enzyme-bound Mn2+ in the presence of H2(17)O indicates that three of the coordination sites on the metal are occupied by water. Sequence alignments, three-dimensional structures, and mechanistic considerations suggest that FosA is related to at least two other metalloenzymes, glyoxalase I and the Mn2+- or Fe2+-containing extradiol dioxygenases. The mechanistic imperative driving the evolution of this previously unidentified superfamily of metalloenzymes is proposed to be bidentate coordination of a substrate or intermediate to the metal center in the enzyme-catalyzed reactions.
苏亚雷斯及其同事最初报道的赋予对抗生素磷霉素[（1R，2S）-1,2-环氧丙基膦酸]抗性的酶[阿雷亚，P.，哈迪松，C.，&苏亚雷斯，J.E.（1990年）《抗菌剂与化疗》34卷，844 - 848页]在本研究中被证明是一种金属谷胱甘肽转移酶。脱辅酶是由16 kDa亚基组成的二聚体。电子顺磁共振光谱和水质子核磁共振纵向弛豫率表明，每个亚基含有一个单核Mn2 +中心，该中心与底物磷霉素强烈相互作用（解离常数Kd = 17 μM），与产物的相互作用较弱（Kd = 1.1 mM），与谷胱甘肽的相互作用非常弱或根本不相互作用。在H2（17）O存在下，酶结合的Mn2 +的电子顺磁共振信号的不均匀展宽表明金属上的三个配位位点被水占据。序列比对、三维结构和机理分析表明，FosA与至少另外两种金属酶有关，即乙二醛酶I和含Mn2 +或Fe2 +的间位二醇双加氧酶。有人提出，驱动这个以前未被识别的金属酶超家族进化的机理要求是在酶催化反应中底物或中间体与金属中心的双齿配位。
