We use a variety of approaches including site-specific mutagenesis and structure determination using x-ray diffraction methods to probe the mechanism of catalysis of nitrogenases, hydrogenases, and select carboxylases. Hydrogenases are complex metalloenzymes that catalyze reversible hydrogen oxidation important for energy metabolism in large number of microbial systems. In collaborative DOE funded work with Joan Broderick's research group we are delineating the enzymatic pathways and mechanism of complex cluster biosynthesis with an emphasis on the role of radical SAM enzyme catalysis in [FeFe]-hydrogenase H cluster biosynthesis.In work funded by DOD-AFOSR we are examining oxygen sensitivity of hydrogenases toward engineering hydrogenases greater tolerance to catalysis in air and in oxygenic phototrophs. Research on the nitrogenase focuses on probing electron gating and substrate binding through capturing mechanistically relevant intermediate states. In DOE funded studies we are also examining functional and structural aspects novel carboxylating enzymes involved in the metabolism of alkenes and ketones.
We are examining the patterns of gene expression associated with a number of different processes. Our work on biological nitrogen fixation focuses on examining metal limitations and alternative nitrogenase expression. In addition we are examining various oxygen protections mechanisms that allow the oxygen sensitive nitrogen reduction process to occur in strict aerobes. These lay the groundwork for a recently NSF funded project to engineer synthetic symbiotic nitrogen fixation to broaden the number of crop plants that can form symbiotic relationships toward the reduction of the application of nitrogenous fertilizers. There is little know about the unique pathways for alkene and ketone metabolism that allow a small group of organisms to grow using these compounds as their sole carbon source. As a component of our DOE funded work on carboxylating enzymes we are examining the links between alkene and ketone metabolism and the central metabolism through transcriptomics experiments. We are using a variety of model organisms to analyze patterns of gene expression associated with metal tolerance and detoxification and oxidative stress as well as biofuel production.
Much of our work in the area of microbial ecology is focused on establishing parameters that constrain the occurrence of life. These studies have been funded by NASA and are aimed at contributing to establishing parameters for habitability for space exploration and the search for life beyond Earth. These studies have been very fundamental in nature asking both what environmental factors constrain the occurrence of various forms of life but also determining the primary environmental underpinnings that control community structure and phylogenetic relatedness mainly in extreme environments. We then relate the results of this work to generate predictive models for the presence of various metabolic processes as a function of environment. We have been exploring a variety of different processes in several different primary field sites. We focus on hydrogen metabolism, nitrogen fixation, methanogenesis, and photosynthesis in field sites in Yellowstone National Park, the Great Salt Lake, the Great Basin in Nevada, and Robertson Glacier in the Canadian Rockies. In work funded by the NSF-PIRE program we are coupling this work in a comparative manner to the characterization of hot springs in the Tengchong region of southern China.
Recently we have developed a keen interest in the evolution of complex biological processes. Nitrogen fixation, hydrogen metabolism, methanogenesis, and photosynthesis are all fairly complicated biochemical processes each requiring enzymes with elaborate cofactors to function. We have been incorporating aspects of cofactor biosynthesis to gain further insights into the evolution of these processes. We have presented results recently challenging established paradigms for the evolution of biological nitrogen fixation. We are exploiting the concatenation of multiple loci and genetic events such as gene duplications and fusions to generate more robust phylogenetic analysis and resulting phylogenetic trajectories. We are exploring the relationship between the nitrogenase components and paralogous gene products involved in chlorophyll/bacteriochlorophyll biosynthesis and methanogenesis. We are exploring the evolution of hydrogenases and probing the ancestry and relationship between [FeFe]-hydrogenases and Nar1p proteins involved in eukaryotic irons sulfur biosynthesis/repair as well as the ancestry of [NiFe]-hydrogenases and electron bifurcation mechanisms and the evolutionary origin of respiration. We are also very interested in the evolutionary relationship between the biosynthetic pathways for coenzyme M involved in methanogenesis and alkene metabolism.
The lab is very basic science driven but many of our key research problems have direct and indirect biotechnological relevance. Our interest in hydrogen production and photosynthesis is very relevant to the biosolar energy solutions such as biohydrogen and the production of lipid based biofuels. Some of this work involves bioprospecting where we are letting nature guide us to superior biotechnological solutions by prospecting in environments that exert natural selective pressures for desired characteristics. In work supported by DOD-AFOSR we are prospecting for more oxygen tolerant hydrogenases by examining environments that cycle from oxic to anoxic over a day night cycle. These phenomena occur in dense phototroph microbial mats or in the water column of stable bodies of water. Bioprospecting in this work is the groundwork for biochemical studies that are aimed at defining the structural determinants for enhanced oxygen tolerance that can be used to engineer more robust hydrogen producing oxygenic phototrophs. Our gene expression work on nitrogen fixation creates a blue print for engineering nitrogen fixation in more robust plant root colonizers promoting more universal associative symbiotic relationships with crop plants to reduce the need for nitrogenous fertilizers in agriculture.