2007 Fiscal Year Final Research Report Summary
Functional proteomics and metabolomics for enzyme discovery in E. coli
| Project/Area Number |
18510177
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Applied genomics
|
|---|
| Research Institution | Keio University |
|---|
Principal Investigator |
ROBERT Martin Keio University, Graduate School of Media and Governance, Assistant Professor (90365487)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SAITO Natsumi Keio University, Graduate School of Media and Governance, Assistant Professor (50287546)
|
|---|
| Project Period (FY) |
2006 – 2007
|
| Keywords | proteomics / metabolomics / functional genomics / analytical chemistry / enzyme / biotechnology / genome |
|---|
| Research Abstract |
Close to one half of the genes in the E. coli bacterium have no experimentally confirmed function. This project was thus aimed at discovering novel enzymes among the uncharacterized E. calf proteome. Multiple proteins were analyzed using bioinformatics tools and databases to select candidates having enzyme-like properties such as containing conserved catalytic motifs or binding domains for common co-factors. We used unbiased and generic in vitro assays to screen uncharacterized proteins for enzymatic activity. The candidates were assayed in vitro using recombinant proteins and a complex and undefined mixture of metabolites. The presence of enzymatic activity was monitored by capillary electrophoresis mass spectrometry (CE-MS) analysis of mixtures. The method allows to both detect unknown activities and identify the substrate(s) and product(s) of the reaction at the same time.
Using this approach several candidate activities were identified. Two were specifically confirmed, including a n
… More
ew kinase and dehydrogenase activity. So far only the ATP/ADP components of the kinase reaction have been observed directly. The actual substrate has yet to be identified and may require the use of alternative analytical methods (e.g. LC-MS etc.). The novel dehydrogenase enzyme was found to display activity on various substrates in vitro but mainly reversibly reduced short chain aldehydes such as succinic semialdehyde or methylglyoxal and produce the respective alcohols. This activity required NADH as electron donor. Confirmation of the exact physiological substrate is continuing using E. call strains carrying a disruption of the dehydrogenase gene or overexpressing its protein. An additional more focused assay method is also being developed and used to match uncharacterized proteins to specific missing enzymatic reactions ("orphan activities") in the E. coli metabolic network.
In summary, we developed a functional proteomics/metabolomics discovery platform and used it to successfully discover several novel enzymatic activities including a novel dehydrogenase possibly involved in stress response in E. coli. The method is highly generic, simple, and can be applied to any organism. Less
|
