3N1Z

X-ray Crystal Structure of Toluene/o-Xylene Monooxygenase Hydroxylase T201S Mutant

Summary for 3N1Z

• Entry DOI: 10.2210/pdb3n1z/pdb
• Related: 1T0Q 2INC 2IND 2RDB 3N1X 3N1Y 3N20
• Descriptor: Toluene o-xylene monooxygenase component, GLYCEROL, HEXAETHYLENE GLYCOL, ... (7 entities in total)
• Functional Keywords: diiron, 4-helix bundle, carboxylate bridge, metalloenzyme, oxidoreductase
• Biological source: Pseudomonas sp.; More
• Total number of polymer chains: 3
• Total formula weight: 107092.74

Authors

McCormick, M.S.,Sazinsky, M.H.,Lippard, S.J. (deposition date: 2010-05-17, release date: 2010-10-13, Last modification date: 2023-09-06)

Primary citation

Song, W.J.,McCormick, M.S.,Behan, R.K.,Sazinsky, M.H.,Jiang, W.,Lin, J.,Krebs, C.,Lippard, S.J.
Active Site Threonine Facilitates Proton Transfer during Dioxygen Activation at the Diiron Center of Toluene/o-Xylene Monooxygenase Hydroxylase.
J.Am.Chem.Soc., 132:13582-13585, 2010

PubMed Abstract: Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was explored by stopped-flow UV-vis and Mössbauer spectroscopy. These studies demonstrate that the dioxygen activation mechanism is preserved in all T201 variants; however, both the formation and decay kinetics of a peroxodiiron(III) intermediate, T201(peroxo), were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O(2) activation in the T201S, T201C, and T201G variants under various reaction conditions revealed that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. A mechanism is postulated for dioxygen activation, and possible structures of oxygenated intermediates are discussed.

PubMed: 20839885
DOI: 10.1021/ja1063795

Experimental method

X-RAY DIFFRACTION (2.9 Å)