Li Jian-Jun, Li Chen, Blindauer Claudia A, Bugg Timothy D H
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
Biochemistry. 2006 Oct 17;45(41):12461-9. doi: 10.1021/bi0612519.
C-C hydrolase enzymes MhpC and BphD catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid and Burkholderia xenovorans LB400 biphenyl degradation pathways and are both members of the alpha/beta-hydrolase family containing a Ser-His-Asp catalytic triad. The catalytic mechanism of this family of enzymes is thought to proceed via a gem-diol reaction intermediate, which has not been observed directly. Site-directed single mutants of BphD in which catalytic residues His-265 and Ser-112 were replaced with Ala were found to possess 10(4)-fold reduced k(cat) values, and in each case, the C-C cleavage step was shown by pre-steady-state kinetic analysis to be rate-limiting. The processing of a 6-(13)C-labeled aryl-containing substrate by these H265A or S112A mutant BphD enzymes was monitored directly by (13)C NMR spectroscopy. A new line-broadened signal was observed at 128 ppm for each enzyme, corresponding to the proposed gem-diol reaction intermediate, over a time scale of 1-24 h. A similar signal was observed upon incubation of the (13)C-labeled substrate with an H114A MhpC mutant, which is able to accept the 6-phenyl-containing substrate, on a shorter time scale. The direct observation of a gem-diol intermediate provides further evidence that supports a general base mechanism for this family of enzymes.
C-C水解酶MhpC和BphD催化大肠杆菌苯丙酸降解途径和洋葱伯克霍尔德菌LB400联苯降解途径上间位环裂变中间体的水解C-C裂解，它们都是含有Ser-His-Asp催化三联体的α/β-水解酶家族成员。该酶家族的催化机制被认为是通过偕二醇反应中间体进行的，但尚未直接观察到。将催化残基His-265和Ser-112替换为Ala的BphD定点单突变体的k(cat)值降低了10^4倍，并且在每种情况下，通过预稳态动力学分析表明C-C裂解步骤是限速步骤。通过^13C NMR光谱直接监测这些H265A或S112A突变体BphD酶对6-(13)C标记的含芳基底物的处理。在1-24小时的时间范围内，每种酶在128 ppm处观察到一个新的线宽信号，对应于所提出的偕二醇反应中间体。在较短的时间范围内，将^13C标记的底物与能够接受含6-苯基底物的H114A MhpC突变体一起孵育时，观察到类似的信号。偕二醇中间体的直接观察提供了进一步的证据，支持该酶家族的一般碱机制。
