The Library
Evidence for a gem-diol reaction intermediate in bacterial C-C hydrolase enzymes BphD and MhpC from C-13 NMR spectroscopy
Tools
Tools
Tools
Li, Jian-Jun, Li, Chen, Blindauer, Claudia A. and Bugg, Tim (2006) Evidence for a gem-diol reaction intermediate in bacterial C-C hydrolase enzymes BphD and MhpC from C-13 NMR spectroscopy. BIOCHEMISTRY, 45 (41). pp. 12461-12469. doi:10.1021/bi0612519
Research output not available from this repository, contact author.
Official URL: http://dx.doi.org/10.1021/bi0612519
Abstract
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-C-13-labeled aryl-containing substrate by these H265A or S112A mutant BphD enzymes was monitored directly by C-13 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 C-13-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.
| Item Type: | Journal Article | ||||
|---|---|---|---|---|---|
| Subjects: | Q Science > QD Chemistry | ||||
| Journal or Publication Title: | BIOCHEMISTRY | ||||
| Publisher: | AMER CHEMICAL SOC | ||||
| ISSN: | 0006-2960 | ||||
| Official Date: | 17 October 2006 | ||||
| Dates: |
|
||||
| Volume: | 45 | ||||
| Number: | 41 | ||||
| Number of Pages: | 9 | ||||
| Page Range: | pp. 12461-12469 | ||||
| DOI: | 10.1021/bi0612519 | ||||
| Publication Status: | Published |
Data sourced from Thomson Reuters' Web of Knowledge
Request changes or add full text files to a record
Repository staff actions (login required)
 |
View Item |
