Ku Shao Yang, Yip Patrick, Howell P Lynne
Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, and Department of Biochemistry, Faculty of Medicine, University of Toronto, Ontario M5G 1X8, Canada.
Acta Crystallogr D Biol Crystallogr. 2006 Jul;62(Pt 7):814-23. doi: 10.1107/S0907444906019895. Epub 2006 Jun 20.
Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.
已从大肠杆菌中分离出5'-磷酸吡哆醛（PLP）依赖性色氨酸酶，并测定了其晶体结构。该结构与普通变形杆菌色氨酸酶和酪氨酸-苯酚裂解酶具有相同的折叠方式和相似的四级结构，但与这两种酶相比，它处于封闭构象。以无辅基形式解析的色氨酸酶结构，其活性位点中没有共价结合的PLP，而是有两个硫酸根离子。硫酸根离子占据PLP的磷酸基结合位点和天然底物色氨酸的α-羧基结合位点。其中一个硫酸根离子与蛋白质的转移酶和PLP结合结构域都有广泛的相互作用，似乎负责将酶保持在其封闭构象。基于硫酸根密度和普通变形杆菌酶的结构，将PLP和底物色氨酸模拟到活性位点中。所得模型与Arg419在使底物定向于PLP并酸化底物的α-质子以进行β-消除中的作用、Lys269在PLP醌型中间体的形成和分解中的作用、Arg230在使底物-PLP中间体定向于催化的最佳构象中的作用以及His463和Tyr74在确定底物特异性中的作用一致，并表明在结构中观察到的封闭构象可能是由底物结合诱导的，并且在催化过程中发生了显著的构象变化。提出了色氨酸酶的催化机制。由于大肠杆菌色氨酸酶多年来一直难以形成高质量衍射晶体，因此已对色氨酸酶的分子表面进行了各种晶体形式的分析，并合理推测在蛋白质的平坦表面上会发生强烈的晶体接触，并且晶体接触表面的大小似乎与晶体的衍射质量相关。
