3ge8 › Citations

Role for threonine 201 in the catalytic cycle of the soluble diiron hydroxylase toluene 4-monooxygenase.

Elsen NL, Bailey LJ, Hauser AD, Fox BG
Biochemistry 48 3838-46 (2009)
Cited: 12 times
EuropePMC logo PMID: 19290655

Abstract

The active site residue Thr-201 in toluene 4-monooxygenase hydroxylase (T4moH) has a structural counterpart in the active sites of all diiron monooxygenases. Thus, our previous finding that mutation of this residue to Ala, Gly, or Ser had no impact on steady-state catalysis or coupling was surprising. In this work, we provide kinetic, biochemical, and structural evidence that one role of Thr-201 may be to stabilize a peroxo-level intermediate during enzyme catalysis. During reactions in the absence of substrate, T201 T4moH slowly consumed O(2) but only a negligible amount of H(2)O(2) was released. In contrast, T201A T4moH gave stoichometric release of H(2)O(2) during reaction in the absence of substrate. Both enzyme isoforms were tightly coupled during steady-state catalysis with saturating toluene and other optimal substrates and exhibited near-identical kinetic parameters. However, rapid mix single-turnover studies showed that T201A T4moH had a faster first-order rate constant for product formation than T201 T4moH did. Comparison of X-ray crystal structures of resting and reduced T201A T4moH in complex with T4moD with comparable structures of T201 T4moHD revealed changes in the positions of several key active site residues relative to the comparable structures of T201 T4moH with T4moD. This combination of catalytic and structural studies offers important new insight into the role of the role of conserved Thr-201, and its contributions to the catalytic reaction cycle.

Articles citing this publication (12)

  1. In-crystal reaction cycle of a toluene-bound diiron hydroxylase. Acheson JF, Bailey LJ, Brunold TC, Fox BG. Nature 544 191-195 (2017)
  2. Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases. Wang W, Liang AD, Lippard SJ. Acc Chem Res 48 2632-2639 (2015)
  3. Insights into the different dioxygen activation pathways of methane and toluene monooxygenase hydroxylases. Bochevarov AD, Li J, Song WJ, Friesner RA, Lippard SJ. J Am Chem Soc 133 7384-7397 (2011)
  4. Active site threonine facilitates proton transfer during dioxygen activation at the diiron center of toluene/o-xylene monooxygenase hydroxylase. Song WJ, McCormick MS, Behan RK, Sazinsky MH, Jiang W, Lin J, Krebs C, Lippard SJ. J Am Chem Soc 132 13582-13585 (2010)
  5. Multiple roles of component proteins in bacterial multicomponent monooxygenases: phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas sp. OX1. Tinberg CE, Song WJ, Izzo V, Lippard SJ. Biochemistry 50 1788-1798 (2011)
  6. Structural basis for biomolecular recognition in overlapping binding sites in a diiron enzyme system. Acheson JF, Bailey LJ, Elsen NL, Fox BG. Nat Commun 5 5009 (2014)
  7. A De Novo Heterodimeric Due Ferri Protein Minimizes the Release of Reactive Intermediates in Dioxygen-Dependent Oxidation. Chino M, Leone L, Maglio O, D'Alonzo D, Pirro F, Pavone V, Nastri F, Lombardi A. Angew Chem Int Ed Engl 56 15580-15583 (2017)
  8. A flexible glutamine regulates the catalytic activity of toluene o-xylene monooxygenase. Liang AD, Wrobel AT, Lippard SJ. Biochemistry 53 3585-3592 (2014)
  9. Mechanistic studies of reactions of peroxodiiron(III) intermediates in T201 variants of toluene/o-xylene monooxygenase hydroxylase. Song WJ, Lippard SJ. Biochemistry 50 5391-5399 (2011)
  10. In Vitro Reconstitution Reveals a Central Role for the N-Oxygenase PvfB in (Dihydro)pyrazine-N-oxide and Valdiazen Biosynthesis. Morgan GL, Li B. Angew Chem Int Ed Engl 59 21387-21391 (2020)
  11. Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase. Oppenheimer M, Pierce BS, Crawford JA, Ray K, Helm RF, Sobrado P. Arch Biochem Biophys 496 123-131 (2010)
  12. Second Coordination Sphere Effects on the Mechanistic Pathways for Dioxygen Activation by a Ferritin: Involvement of a Tyr Radical and the Identification of a Cation Binding Site. Yeh CG, Mokkawes T, Bradley JM, Le Brun NE, de Visser SP. Chembiochem 23 e202200257 (2022)


Quick links