title: The "Gln-Type" Thiol Dioxygenase from Azotobacter vinelandii is a 3-Mercaptopropionic Acid Dioxygenase.
abstract: Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine to produce cysteinesulfinic acid. Bacterial CDOs have been subdivided as either "Arg-type" or "Gln-type" on the basis of the identity of conserved active site residues. To date, "Gln-type" enzymes remain largely uncharacterized. It was recently noted that the "Gln-type" enzymes are more homologous with another thiol dioxygenase [3-mercaptopropionate dioxygenase (MDO)] identified in Variovorax paradoxus, suggesting that enzymes of the "Gln-type" subclass are in fact MDOs. In this work, a putative "Gln-type" thiol dioxygenase from Azotobacter vinelandii (Av) was purified to homogeneity and characterized. Steady-state assays were performed using three substrates [3-mercaptopropionic acid (3mpa), l-cysteine (cys), and cysteamine (ca)]. Despite comparable maximal velocities, the "Gln-type" Av enzyme exhibited a specificity for 3mpa (kcat/KM = 72000 M(-1) s(-1)) nearly 2 orders of magnitude greater than those for cys (110 M(-1) s(-1)) and ca (11 M(-1) s(-1)). Supporting X-band electron paramagnetic resonance (EPR) studies were performed using nitric oxide (NO) as a surrogate for O2 binding to confirm obligate-ordered addition of substrate prior to NO. Stoichimetric addition of NO to solutions of 3mpa-bound enzyme quantitatively yields an iron-nitrosyl species (Av ES-NO) with EPR features consistent with a mononuclear (S = (3)/2) {FeNO}(7) site. Conversely, two distinct substrate-bound conformations were observed in Av ES-NO samples prepared with cys and ca, suggesting heterogeneous binding within the enzymatic active site. Analytical EPR simulations are provided to establish the relative binding affinity for each substrate (3map > cys > ca). Both kinetic and spectroscopic results presented here are consistent with 3mpa being the preferred substrate for this enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, College of Sciences, The University of Texas at Arlington , Arlington, Texas 76019, United States.
authors.forename: Brad S
authors.initials: BS
authors.lastname: Pierce
authors.affiliation: Department of Chemistry and Biochemistry, College of Sciences, The University of Texas at Arlington , Arlington, Texas 76019, United States.
authors.forename: Bishnu P
authors.initials: BP
authors.lastname: Subedi
authors.affiliation: Department of Chemistry and Biochemistry, College of Sciences, The University of Texas at Arlington , Arlington, Texas 76019, United States.
authors.forename: Sinjinee
authors.initials: S
authors.lastname: Sardar
authors.affiliation: Department of Chemistry and Biochemistry, College of Sciences, The University of Texas at Arlington , Arlington, Texas 76019, United States.
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Crowell
mesh_terms: D015097:3-Mercaptopropionic Acid / Q000737:chemistry*; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain; D049308:Dioxygenases / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005998:Glycine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation
chemical_list: D015097:3-Mercaptopropionic Acid; D049308:Dioxygenases; D005998:Glycine