Dept. of Microbiology and
Harvard Medical School
HIM, Room 1047
4 Blackfan Circle
Boston, MA 02115
Beckwith Lab Home
The mechanism of protein disulfide bond formation:
An important role for cysteines in many proteins is to provide ways of generating covalent linkages (disulfide bonds) that add stability to many exported and cell envelope proteins in bacteria. We have defined two enzymes (DsbA and DsbB) involved in disulfide bond formation in the bacterium Escherichia coli and characterized the mechanisms of action of these enzymes. We have analyzed to other bacteria and discovered a novel pathway in certain groups of bacteria, including Mycobacterium tuberculosis. These latter bacteria have DsbA, but use a different enzyme from DsbB in disulfide bond formation. This enzyme is a homologue of a human enzyme involved in blood clotting, vitamin K epoxide reductase (VKOR).
Evolution of novel pathways for disulfide bond formation and reduction: Using mutants of E. coli that are deleted for genes encoding DsbA or DsbB, and, therefore, cannot make protein disulfide bonds, we have evolved bacteria to utilize alternative pathways for this process. This has revealed numerous ways in which mutants can generate alternatives. We have also generated mutants that are defective in the cytoplasmic pathways of E. coli that are needed to reduce the cysteines of important enzymes such as ribonucleotide reductase. Again, we have evolved E. coli to generate novel pathways of reduction. In both these studies, we discover new potential functions of proteins and reveal an extraordinary evolutionary plasticity of the bacteria.
Inhibitors of the disulfide bond-forming pathway as potential antibiotics: We have developed a highly sensitive assay for the formation of disulfide bonds in E. coli. Since the VKOR of M. tuberculosis and the DsbBs of bacteria such as the Pseudomonads, Acinetobacter and Klebsiella all will complement an E. coli mutant lacking DsbB, we can do high throughput screening with these various foreign proteins acting in E. coli. A number of compounds obtained already may be useful for antibiotic development and also for studying the mechanisms of action of these enzymes.
Landeta, C., McPartland, L., Tran, Z.,Tanweer, T., Minami, S., Rock, J., Meehan, B.M. Kim, T. Balasubramanian, D. Pinkham, J., Audette, R., Toosky, M., Rubin, E.J. Lory,S., Pier, G., Boyd, D., and Beckwith, J.. “Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes” Molecular Microbiology” (2019 in press).
Ke N, Landeta C, Wang X, Boyd D, Eser M, and Beckwith J. Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation. J Bacteriol. 2018 Jul 25;200(16). pii: e00137-18. doi: 10.1128/JB.00137-18. Print 2018 Aug 15.
Hibender, S., Landeta, C., Berkmen M., Beckwith, J. and, Boyd, D. Aeropyrum pernix membrane topology of protein VKOR promotes protein disulfide bond formation in two subcellular compartments. Microbiology, 2017 Nov 15. doi: 10.1099/mic.0.000569.
Beckwith, J., and Pierce, R. Genes and Human Behavior: Ethical Implications in Gerlai, R. and Crusio, W., Editors. Molecular-Genetic and Statistical Techniques for Behavioral and Neural Research. (Elsevier:2018). Chapter: 25.
Beckwith, J., Bergman, K., Carson, M., Doerr, T., Geller, L., Pierce, R., Krimsky, S., Martin, C., Santiago, M., VAshlishan, Murray, A., Warren, C., Zichterman, C. Using Dialogues to Explore Genetics, Ancestry and Race. Am. Biol. Teacher. 79:525-537 (2017).