3-Mercaptopropionic acid dioxygenase (MDO) isolated from the soil bacteria Azotobacter vinelandii is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of 3-mercaptopropionic acid (3mpa) to produce 3-sulfinopropionic acid (3spa). With one exception, the 3-His facial triad and outer coordination sphere residues within the MDO active site are equivalent to what is observed for the more extensively characterized cysteine dioxygenase (CDO). Specifically, the CDO Arg-residue involved in electrostatic stabilization of the substrate is replaced by Gln (Q67) in MDO. In this work the pH dependent steady-state kinetics, proton-inventory and viscosity effects are utilized to investigate substrate-specificity and substrate-coordination within the MDO active site. Complementary X-band EPR studies were performed using nitric oxide as a surrogate for O2-binding. Despite the largely conserved active site configuration among these enzymes, MDO exhibits greater substrate promiscuity as compared to CDO. Indeed, comparable maximal velocities and coupling efficiencies are observed in reactions performed using three substrates [3-mercaptopropionic acid (3mpa), L-cysteine (cys) and cysteamine (ca)]. However, the efficiency (kcat/KM) of MDO is several orders of magnitude greater in reactions utilizing 3mpa as a substrate (relative to cys and ca). Thus establishing that 3mpa is the preferred substrate for this enzyme. EPR and Mössbauer results suggest that coordination of the substrate to the MDO Fe-site occurs via the substrate thiolate group only. By contrast, cys-coordination to the CDO Fe-site is bidentate via thiolate and neutral amine. This coordination difference likely explains the relaxed substrate-specificity observed for MDO.

Kinetic and spectroscopic interrogation of a promiscuous thiol dioxygenase: 3-mercaptopropionic acid dioxygenase

251st National Meeting (2016)

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