2015 Beckman Symposium   

Isaac Larkin

Presentation Date:

Isaac Larkin

28 - Probing the structure and function of a Carbon monoxide oxidase-like protein in Caulobacter crescentus

University of Chicago

Biochemistry and Molecular Biology

Aerobic carbon monoxide oxidase (COX) is an oxygen-stable enzyme that enables some bacteria to use carbon monoxide as an energy and a carbon source (1), and has the potential to contribute to the conversion of syngas, the mixture of CO, H2 and CO2 that is the product of organic waste gasification, into useful organic carbon (2). However, bacteria with COX genes tend to be extremophiles or grow slowly (Td >3 h), and thus are not ideal vehicles for biotechnological innovation (3, 4). We found genes, CCNA_00021 and 00022, that are predicted by BLAST homology to code for the Large and Small subunits of a COX in Caulobacter crescentus, an extensively studied model for cell cycle regulation that is both genetically malleable and relative fast growing (5, 6). To investigate these genes, we analyzed transcriptional data and determined that they are upregulated during carbon starvation. To determine whether C. crescentus can consume CO as an energy or carbon source, we performed growth curves of wild type C. crescentus in rich (PYE) and Glucose Exhaustion Media in gas-tight vessels containing different mixtures of air and CO, and then used gas chromatography to analyze the evolution of the vessels€ headspace gas. Our results indicate that while C. crescentus is more tolerant of CO than E. coli, it does not consume CO while growing in either PYE or Glucose Exhaustion Media. We therefore conclude that CCNA_00021 and 00022, though structurally similar to a COX, have a different function. We have determined through €-Galactosidase assays that the genes are upregulated during stationary phase and during carbon starvation, indicating that their function may be important for survival in high stress, low nutrient environments. To investigate this function, we have developed a protocol to inducibly coexpress tagged versions of CCNA_00021 and 00022 with the genes necessary to synthesize the COX€s unstable molybopterin cofactor. We plan to purify the protein and use a colorimetric assay to determine its substrate. Then, we will attempt to crystallize the protein and solve its structure in order to probe its active site. References 1. Holger Dobbek, Lothar Gremer, Ortwin Meyer, and Robert Huber . Crystal structure and mechanism of CO dehydrogenase, a molybdo iron-sulfur flavoprotein containing S-selanylcysteine. Proc National Academy of the Sciences USA. pp. 8884-8889, 1999. 2. Reeves, Andrew and Datta, Rathin. 2012. Genes Encoding Key Catalyzing Mechanisms for Ethanol Production from Syngas Fermentation. U.S. Patent 13/655,054, filed October 18, 2012, and issued April 25, 2013. 3. Paul D, Kumar R, Nanduri B, French T, Pendarvis K, et al. Proteome and Membrane Fatty Acid Analyses on Oligotropha carboxidovorans OM5 Grown under Chemolitho-autotrophic and Heterotrophic Conditions. PLoS ONE 6(2): e17111, 2010. 4. Dimitry Sorokin, Tatjana Tourova, Olga Kovaleva, et al. Aerobic carboxydotrophy under extremely haloalkaline conditions in Alkalispirillum/Alkalilimnicola strains isolated from Soda Lakes. Microbiology pp 819-827, 2010. 5. Jigar Patel, Qiong Zhang, R. Michael L. McKay, et al. Genetic Engineering of Caulobacter crescentus for Removal of Cadmium from Water. Applied Biochemistry and Biotechnology, V. 160, pp 232-243, 2010. 6. Alison K. Hottes, Maliwan Meewan, Desiree Yang, et al. Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media. Journal of Bacteriology, V. 186, pp 1448-1461, 2004.

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