Alahuhta Markus, Taylor Larry E, Brunecky Roman, Sammond Deanne W, Michener William, Adams Michael W W, Himmel Michael E, Bomble Yannick J, Lunin Vladimir
BioSciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
Acta Crystallogr D Biol Crystallogr. 2015 Sep;71(Pt 9):1946-54. doi: 10.1107/S1399004715013760. Epub 2015 Aug 25.
The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase (PL3) enzyme has been thoroughly characterized using a series of point mutations, X-ray crystallography, pK(a) calculations and biochemical assays. The X-ray structures of seven PL3 active-site mutants, five of them in complex with intact trigalacturonic acid, were solved and characterized structurally, biochemically and computationally. The results confirmed that Lys108 is the catalytic base, but there is no clear candidate for the catalytic acid. However, the reaction mechanism can also be explained by an antiperiplanar trans-elimination reaction, in which Lys108 abstracts a proton from the C5 atom without the help of simultaneous proton donation by an acidic residue. An acidified water molecule completes the anti β-elimination reaction by protonating the O4 atom of the substrate. Both the C5 hydrogen and C4 hydroxyl groups of the substrate must be orientated in axial configurations, as for galacturonic acid, for this to be possible. The wild-type C. bescii PL3 displays a pH optimum that is lower than that of Bacillus subtilis PL1 according to activity measurements, indicating that C. bescii PL3 has acquired a lower pH optimum by utilizing lysine instead of arginine as the catalytic base, as well as by lowering the pK(a) of the catalytic base in a unique active-site environment.
利用一系列点突变、X射线晶体学、pK(a)计算和生化分析，对嗜热栖热放线菌（Caldicellulosiruptor bescii）家族3果胶酸裂解酶（PL3）的独特活性位点进行了全面表征。解析并从结构、生化和计算方面对7个PL3活性位点突变体的X射线结构进行了表征，其中5个与完整的三半乳糖醛酸形成复合物。结果证实，Lys108是催化碱，但没有明确的催化酸候选者。然而，反应机制也可以用反式共平面反式消除反应来解释，即Lys108从C5原子夺取一个质子，而无需酸性残基同时供质子的帮助。一个酸化的水分子通过使底物的O4原子质子化来完成反β消除反应。底物的C5氢和C4羟基都必须以轴向构型排列，就像半乳糖醛酸那样，才有可能发生这种情况。根据活性测量，野生型嗜热栖热放线菌PL3的最适pH低于枯草芽孢杆菌PL1，这表明嗜热栖热放线菌PL3通过利用赖氨酸而非精氨酸作为催化碱，并在独特的活性位点环境中降低催化碱的pK(a)，从而获得了较低的最适pH。
