Li Shengying, Ouellet Hugues, Sherman David H, Podust Larissa M
Life Sciences Institute, Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2009 Feb 27;284(9):5723-30. doi: 10.1074/jbc.M807592200. Epub 2009 Jan 4.
The cytochrome P-450 PikC from Streptomyces venezuelae exhibits significant substrate tolerance and performs multiple hydroxylation reactions on structurally variant macrolides bearing the deoxyamino sugar desosamine. In previously determined co-crystal structures (Sherman, D. H., Li, S., Yermalitskaya, L. V., Kim, Y., Smith, J. A., Waterman, M. R., and Podust, L. M. (2006) J. Biol. Chem. 281, 26289-26297), the desosamine moiety of the native substrates YC-17 and narbomycin is bound in two distinct buried and surface-exposed binding pockets, mediated by specific interactions between the protonated dimethylamino group and the acidic amino acid residues Asp(50), Glu(85), and Glu(94). Although the Glu(85) and Glu(94) negative charges are essential for maximal catalytic activity of native enzyme, elimination of the surface-exposed negative charge at Asp(50) results in significantly enhanced catalytic activity. Nevertheless, the D50N substitution could not rescue catalytic activity of PikC(E94Q) based on lack of activity in the corresponding double mutant PikC(D50N/E94Q). To address the specific role for each desosamine-binding pocket, we analyzed the x-ray structures of the PikC(D50N) mutant co-crystallized with narbomycin (1.85A resolution) and YC-17 (3.2A resolution). In PikC(D50N), the desosamine moiety of both YC-17 and narbomycin was bound in a catalytically productive "buried site." This finding suggested a two-step substrate binding mechanism, whereby desosamine is recognized in the two subsites to allow the macrolide substrate to sequentially progress toward a catalytically favorable orientation. Collectively, the binding, mutagenesis, kinetic, and x-ray structural data suggest that enhancement of the catalytic activity of PikC(D50N) is due to the facilitated relocation of substrate to the buried site, which has higher binding affinity, as opposed to dissociation in solution from the transient "surface-exposed site."
委内瑞拉链霉菌的细胞色素P-450 PikC表现出显著的底物耐受性，并且能对带有脱氧氨基糖地索胺的结构多样的大环内酯类化合物进行多种羟基化反应。在先前测定的共晶体结构中（谢尔曼，D. H.，李，S.，叶尔马利茨卡娅，L. V.，金，Y.，史密斯，J. A.，沃特曼，M. R.，以及波杜斯特，L. M.（2006年）《生物化学杂志》281卷，26289 - 26297页），天然底物YC - 17和纳罗霉素的地索胺部分结合在两个不同的埋藏和表面暴露的结合口袋中，这是由质子化的二甲基氨基与酸性氨基酸残基天冬氨酸（50）、谷氨酸（85）和谷氨酸（94）之间的特异性相互作用介导的。尽管谷氨酸（85）和谷氨酸（94）的负电荷对于天然酶的最大催化活性至关重要，但消除天冬氨酸（50）处表面暴露的负电荷会导致催化活性显著增强。然而，基于相应的双突变体PikC（D50N/E9Q）缺乏活性，D50N取代无法挽救PikC（E9CQ）的催化活性。为了阐明每个地索胺结合口袋的具体作用，我们分析了与纳罗霉素（分辨率为1.85埃）和YC - 17（分辨率为3.2埃）共结晶的PikC（D50N）突变体的X射线结构。在PikC（D50N）中，YC - 17和纳罗霉素的地索胺部分都结合在一个具有催化活性的“埋藏位点”中。这一发现提示了一种两步底物结合机制，即地索胺在两个亚位点中被识别，以使大环内酯类底物能够依次朝着催化有利的方向进展。总体而言，结合、诱变、动力学和X射线结构数据表明，PikC（D50N）催化活性的增强是由于底物更容易重新定位到具有更高结合亲和力的埋藏位点，而不是在溶液中从短暂的“表面暴露位点”解离。
