Processive enzymes conduct much of the cellulose and chitin turnover in the biosphere, and are thus important contributors to the global carbon and nitrogen cycles and the major components in industrial cocktails in biomass conversion.  However, the mechanism by which processive enzymes deconstruct their polysaccharide substrates is not understood at the molecular level. We approach this problem using molecular dynamics and free energy calculations to examine the roles of active site features in well-characterized processive chitinases from the bacterium Serratia marcescens. These molecular-level insights complement ongoing experimental work by our collaborators at the Norwegian University of Life Sciences.  Our most recent discovery includes identification of three dynamical hallmarks of processivity in glycoside hydrolases. Findings from the chitinase/chitin model systems are directly relevant to our understanding of cellulase/cellulose systems and may significantly impact the way we approach biotechnology development for enzymatic conversion of biomass to biofuels.

Glycoside hydrolase catalytic domains are a significant focus of much of the ongoing biomass conversion protein engineering research, with a clearly defined functional role of glycosidic bond cleavage as a straightforward target for improvement. Recently, the research community has begun to recognize the additional potential of harnessing the carbohydrate recognition capabilities of carbohydrate binding modules (CBMs) for more effective biomass conversion or multitudes of other biotechnological applications. As a result, many fundamental questions remain as to how these proteins recognize carbohydrate substrates. We are currently using molecular modeling to uncover the mechanisms behind oligomeric and amorphous carbohydrate recognition in Type B CBMs. We are also interested in how the coupling of tandem CBMs contributes to cooperative binding and avidity.

Conventional hydrodesulfurization is an appropriate technology for large-scale removal of sulfur from refined products. However, it is largely ineffective at removing the refractory thiophenic compounds that make up the majority of sulfur in finished fuels. Biodesulfurization is a complementary low-temperature and pressure desulfurization route using bacterial biocatalysts to cleave C-S bonds without degrading fuel energy density. Commercialization of biodesulfurization hinges upon overcoming key technological challenges associated with protein stability, activity, and specificity. Our group uses  computational techniques to uncover the molecular-level mechanisms of a Rhodococcus erythropolis desulfinase. These insights serve as a platform for developing and validating a rational design strategy for desulfinase biotechnology.

Bioremediation has been increasingly implemented as a safe and cost-effective in situ approach in the degradation of environmental pollutants. Membrane-based technologies that immobilize biocatalysts offer the ability to retain and reuse the catalytic components. The use of isolated enzymes over microorganisms provides additional advantages including reduced generation of toxic byproducts, enhanced bioavailability through use of organic co-solvents or surfactants, and the possibility of large-scale production through recombinant DNA technology. With Dr. Edith Glazer, our group is investigating development of cytochrome P450BM3 variants for inclusion in such an enzymatic remediation technology. P450BM3, a fatty acid hydroxylase, was chosen for its high production yield, tunable reaction profile, and excellent enzymatic efficiency. Despite these advantages, obtaining optimal performance necessitates considering extending the range of substrate specificity and potential enhancements to withstand extreme conditions. By combining molecular simulation and biochemical characterization, we elucidate the factors governing these properties enabling design of high reactivity, high stability variants. 

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YKL-40 is a mammalian glycoprotein that has been implicated as a biomarker associated with progression, severity, and prognosis of chronic inflammatory diseases and a multitude of cancers. Though the association of YKL-40 with physical maladies is well-known, both the function and conclusive identification of physiological ligand of this enzyme remains elusive. Speculation as to its function varies from both inhibiting and antagonizing collagen fibril formation as a result of injury or disease, as well as inferring drug resistance and increasing cell migration leading to progression of cancer, and protection from chitin-containing pathogens. Understanding the mechanism by which YKL-40 binds known ligands can help identify potential treatment therapeutics as alternative binding ligands for inactivation.