Description:
<jats:p>Biodesulfurization is a metabolically important process that occurs naturally in bacterial cells found near oil and coal deposits. In sulfur‐specific biodesulfurization, the dsz pathway, enzymes selectively remove the organic sulfur found in crude oil without disrupting the carbon backbone. The rate‐limiting step within this metabolic pathway is the conversion of 2‐(2′‐hydroxyphenyl)benzenesulfinate (HPBS) to 2‐hydroxybiphenyl using HPBS desulfinase (DszB). The DszB enzyme from <jats:italic>Nocardia asteroides</jats:italic> A3H1 was overexpressed in <jats:italic>E. coli,</jats:italic> purified and characterized kinetically. Homology studies identified an active site loop with conserved amino acids that could play a role in the specificity for different organosulfur compounds found in crude oil. Point mutations were generated to study the importance of amino acids 195 and 200 on the specificity of the enzyme. Three mutant enzymes (A195R, A200R and A195/200R) prepared using codon‐optimized gene constructs were co‐expressed with GroESL and purified from <jats:italic>E. coli</jats:italic> using Ni<jats:sup>2+</jats:sup>‐affinity chromatography. Substrate specificity experiments were performed to define the role of the active site loop on specificity. Kinetic measurements indicate a decreased specificity for HPBS. Fixed‐time sulfite assays indicate an increased preference for smaller ringed substrates, demonstrating the importance of amino acids 195 and 200 and defining the specificity of the enzyme.</jats:p><jats:p><jats:bold>Support or Funding Information</jats:bold></jats:p><jats:p>Supported by NSF grant, CHE 1461175, and James Madison University Department of Chemistry and Biochemistry</jats:p><jats:p>This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in <jats:italic>The FASEB Journal</jats:italic>.</jats:p>