Bernat B A, Laughlin L T, Armstrong R N
Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 1999 Jun 8;38(23):7462-9. doi: 10.1021/bi990391y.
The fosfomycin resistance protein FosA is a member of a distinct superfamily of metalloenzymes containing glyoxalase I, extradiol dioxygenases, and methylmalonyl-CoA epimerase. The dimeric enzyme, with the aid of a single mononuclear Mn2+ site in each subunit, catalyzes the addition of glutathione (GSH) to the oxirane ring of the antibiotic, rendering it inactive. Sequence alignments suggest that the metal binding site of FosA is composed of three residues: H7, H67, and E113. The single mutants H7A, H67A, and E113A as well as the more conservative mutants H7Q, H67Q, and E113Q exhibit marked decreases in the ability to bind Mn2+ and, in most instances, decreases in catalytic efficiency and the ability to confer resistance to the antibiotic. The enzyme also requires the monovalent cation K+ for optimal activity. The K+ ion activates the enzyme 100-fold with an activation constant of 6 mM, well below the physiologic concentration of K+ in E. coli. K+ can be replaced by other monovalent cations of similar ionic radii. Several lines of evidence suggest that the K+ ion interacts directly with the active site. Interaction of the enzyme with K+ is found to be dependent on the presence of the substrate fosfomycin. Moreover, the E113Q mutant exhibits a kcat which is 40% that of wild-type in the absence of K+. This mutant is not activated by monovalent cations. The behavior of the E113Q mutant is consistent with the proposition that the K+ ion helps balance the charge at the metal center, further lowering the activation barrier for addition of the anionic nucleophile. The fully activated, native enzyme provides a rate acceleration of >10(15) with respect to the spontaneous addition of GSH to the oxirane.
磷霉素抗性蛋白FosA是一个独特的金属酶超家族的成员，该超家族包括乙二醛酶I、间位二醇双加氧酶和甲基丙二酰辅酶A差向异构酶。这种二聚体酶在每个亚基中单个单核Mn2+位点的帮助下，催化谷胱甘肽（GSH）加到抗生素的环氧乙烷环上，使其失去活性。序列比对表明，FosA的金属结合位点由三个残基组成：H7、H67和E113。单突变体H7A、H67A和E113A以及更保守的突变体H7Q、H67Q和E113Q在结合Mn2+的能力上显著下降，并且在大多数情况下，催化效率和赋予对抗生素抗性的能力也下降。该酶还需要一价阳离子K+以实现最佳活性。K+离子以6 mM的激活常数将酶激活100倍，远低于大肠杆菌中K+的生理浓度。K+可以被其他具有相似离子半径的一价阳离子取代。几条证据表明，K+离子直接与活性位点相互作用。发现该酶与K+的相互作用依赖于底物磷霉素的存在。此外，E113Q突变体在没有K+的情况下表现出的kcat是野生型的40%。该突变体不能被一价阳离子激活。E113Q突变体的行为与K+离子有助于平衡金属中心电荷的观点一致，这进一步降低了阴离子亲核试剂加成的活化能垒。相对于GSH自发加成到环氧乙烷上，完全激活的天然酶提供了>10(15)的速率加速。
