Purification, Characterization, and Active Site Studies of 2-(2'-Hydroxyphenyl) Benzenesulfinate Desulfinase (DSZB) from Nocardia Asteroides Sp. Strain A3H1

Abstract

Dibenzophene (DBT) and its complex heterocyclic derivatives comprise most of the sulfur that contaminates fossil fuels. The 2-(2´-hydroxyphenyl) benzenesulfinate desulfinase (DszB) enzyme metabolize sulfur from DBT compounds. DszB, one of the four enzymes of the desulfurizing pathway, catalyzes the carbon-sulfur bond cleavage in the last, and rate limiting step. The wildtype A3H1-DszB and the recombinant A3H1- R84Q-Y24Fand A3H1-R84Q-C27S DszBs from Nocarida asteroides sp. strain A3H1 were purified and characterized. The A3H1-R84Q-C27S- DszB was inactive. The temperature and pH optima for wildtype and A3H1-R84Q-Y24F DszBs were 35 ˚C and 8.5, respectively; the Km and kcat were 3.15 ± 0.74 µM and 1.24 ± 0.054 min-1, and 51.25 ± 0.96 µM and 4.33 ± 0.67 min-1, respectively. Overall, the A3H1-DszB showed a slower turnover rate and a moderately lower specificity for substrate when compared to the other DszB homologs referenced in this study. The mutant A3H1-R84Q-Y24F-DszB revealed that the enzyme was more susceptible to inhibition than the wildtype. Both DszBs were inhibited by 2,2-biphenol. Significant inhibition by a mixture of HBP and sulfite was observed in both enzymes.