Structural and Mechanistic Insights from High Resolution Crystal Structures of the Toluene-4- Monooxygenase Catalytic Effector Protein, Nad(p)h Oxidase and Choline Oxidase

Single crystals have been obtained of the toluene-4-monooxygenase catalytic effector protein, the SeMet-enriched protein and a truncated isoform missing ten amino acids from the N-terminus. Complete X-ray diffraction data sets have been collected and analyzed to 2.0, 3.0, and 1.96 Å resolution for the native, SeMet and truncated isoform crystals, respectively. The native and SeMet proteins crystallized in space group P6122 (unit cell parameters a = b = 86.41 ± 0.15, c = 143.90 ± 0.27 Å), whereas the truncated isoform crystallized in space group P213 ( a = b = c = 86.70 ± 0.47 Å). Matthews coefficient calculations suggest either two or three molecules per asymmetric unit in the P6122 space group and two molecules per asymmetric unit in the P213 space group. Experimental phases from MAD analysis of the SeMet isoform and molecular replacement of the truncated isoform confirm the presence of two molecules per asymmetric unit in each case. These crystallographic results are the first available for the evolutionary related but functionally diversified catalytic effector proteins from the multicomponent diiron monooxygenase family. 1 Adapted from Orville, A.M., Studts, J.M., Lountos, G.T., Mitchell, K.H., and Fox, B.G. (2005) Crystallization and preliminary analysis of native and N-terminal truncated isoforms of toluene-4monooxygenase catalytic effector protein. Acta Cryst. D59, 572-575.