(627a) Understanding the Role of Conformational Change in Product Inhibition of 2-(2’ hydroxyphenyl)Benzenesulfinate Desulfinase (Dszb

Increasingly stringent regulation of sulfur oxide emissions and environmental stewardship necessitates more effective fossil fuel desulfurization technologies. While traditional catalytic means of desulfurization effectively remove simple sulfur compounds, more complex thiophenic molecules remain intact; these thiophenic molecules now account for the majority of sulfur emissions from liquid transportation fuels. Biodesulfurization via enzyme catalysis enables highly specific, rapid thiophenic desulfurization occurring at ambient temperature and pressure. The 4-step catabolic pathway converts dibenzothiophene (DBT), a common crude oil contaminant, into the sulfur-free molecule 2-hydroxybiphenyl (2-HBP) without the disruption of carbon-carbon bonds. 2-hydroxybiphenyl desulfinase (DszB), the rate-limiting enzyme in this biocatalytic process, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, fundamental understanding of the molecular mechanisms of DszB must be developed. Based on crystallographic evidence, we hypothesize that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipate this conformational change is responsible, in part, for observed product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced and purified via Escherichia coli BW25113 to test these hypotheses. Activity and the conformational changes of DszB in the presence of 2-HBP were evaluated. The activity of recombinant DszB appears comparable to the natively expressed enzyme and is inhibited via competitive binding of the product, 2-HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to 2-HBP; we found that both product and substrate generate similar structural changes. For more time sensitive characterizations, a fusion protein – DszB and a Small Ubiquitin-like Modifier (SUMO Tag) – was created to improve enzyme stability. The resulting fusion protein was then subjected to hydrogen deuterium exchange mass spectroscopy to monitor local changes in protein conformation in response to 2-HBP. Results linking conformational change to kinetic inhibition provide a basis for catalytic improvement of DszB via rational design through identification of groups of residues responsible for inhibitory effects.