Mechanism of radical-mediated dehydration reactions in anaerobic bacteria
Final Report Abstract
This project elucidated the substrate stereochemistry ofthis dehydration. The enzyme catalyses the abstraction oflhe 2-Re and 3-Si protons from 4 as well as the substitution of the hydroxyl group by hydrogen with retention of configuration. This result underpins the proposed modelling of 4 into the crystal structure of 4-hydroxybutyryl-CoA dehydratase. We were able to produce the enzyme in Escherichia coli and generate mutants of the conserved residues around the aclive site with the prosthetic groups [4Fe-4S] cluster and FAD. All mutated amino acids including the three cysteins and the histidine coordinating the [4Fe-4S] cluster as well as two glulamales and one tyrosine are essential for aclivity. Only the YF mutant retained 1% activity of that ofthe wild type. Recently 4-hydroxybutyryl-CoA dehydratase was shown lo act as key player in the 5th cycle of CO2 fixation, the 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea.
Publications
- (2006) Characterisation of (R)-2- hydroxyisocaproaie dehydrogenase and a family III CoA-transferase involved in the reduction of L-leucine to iscocaproate by Clostridium difficile. Appl. Environ. Microbiol. 72, 6062-6069
Kim, J., Darley, D., Selmer, T. & Buckel, W.
- (2006) Radical enzymes in anaerobes. Ann. Rev. Microbiol., 60, 27-49
Buckel, W. & Golding, B. T.
- (2007) 4-Hydroxybutyryl-CoA Dehydratase. In: Handbook of Metalloproteins Online Edition (A. Messerschmidt, ed.) John Wiley & Sons Ltd., Sussex, UK
Martins, B., Messerschmidt, A., Friedrich, P., Zhang, J. & Buckel, W.
(See online at https://doi.org/10.1002/0470028637.met214) - (2007) A 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. Science 318, 1782-1786
Berg, I., Kockelkorn, D., Buckel, W. & Fuchs, G.
- (2007) ATP- and redox-induced conformational changes in the activator of the radical enzyme 2-hydroxyisocaproyl-CoA dehydratase. C. R. Chimie 10, 742-747
Kim, J., Lu, Y. & Buckel, W.
- (2008) An allylic ketyl radical intermediate in clostridial amino acid fermentation. Nature 452, 239-242
Kim, J., Darley, D., Buckel, W. & Pierik, A. J.
- (2008) Der stereochemische Verlauf der enzymatischen Wassereliminierung von 4-Hydroxybutyryl-Coenzym A zu Crotonyl-Coenzym A. Angew. Chem. 120, 3298-3301. The complete stereochemistry of the enzymatic dehydration of 4-hydroxybutyryl-coenzyme A to crotonyl-coenzyme A. Angew. Chem. Int. Ed. 47, 3254-3257
Friedrich, P., Darley, D., Golding, B. T. & Buckel, W.
- (2008) Response to the comment on "A 3-hydroxypropionale/4-hydroxybutyrale autotrophic carbon dioxide assimilation pathway in Archaea." Science 321, 342c
Berg, I., Kockelkom, D., Buckel, W. & Fuchs, G.
- (2008) Spectroscopic evidence for an all-ferrous [4Fe-4S]0 cluster in the super-reduced activator of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans, J. Inorg. Biol. Chem. 13, 563-574
Hans, M., Buckel, W. & Bill, E.
