Inhibition Mechanisms of 2'-Hydroxybiphenyl-2-sulfinate Desulfinase ().

Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2'-Hydroxybiphenyl-2-sulfinate desulfinase (), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. IGTS8 was recombinantly produced and purified via BL21 to test these hypotheses. Activity and the resulting conformational change of in the presence of HBP were evaluated. The activity of recombinant was comparable to the natively expressed enzyme and was inhibited via competitive binding of the product, HBP. Using circular dichroism, global changes in conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of bound to common, yet structurally diverse, crude oil contaminants 2',2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root-mean-square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that the combination of a molecule's hydrophobicity and nonspecific interactions with nearby functional groups contributes to a competitive inhibition mechanism that locks in a closed conformation and precludes substrate access to the active site.