W0400

Crystallization and Structural Analysis of the Toluene-4-monooxygenase Catalytic Effector Protein. George T. Lountos^a, Joey M. Studts^b, Kevin H. Mitchell^b, Brian G. Fox^b_, and Allen M. Orville^a, ^aSchool of Chemistry & Biochemistry, Georgia Inst. of Technology, Atlanta, GA 30332, ^bDeptt of Biochemistry, Univ. of Wisconsin, Madison, WI 53706.

Toluene-4-monooxygenase (T4MO) from Pseudomonas menodocina KR1 is a multicomponent enzyme that catalyzes the NADH- and O₂-dependent hydroxylation of toluene to form para-cresol¹. The T4MO enzyme complex consists of an NADH oxidoreductase, a Rieske-type ferredoxin, a diiron-containing hydroxylase, and a catalytic effector protein (T4MOD). Crystals have been obtained for T4MOD in native, SeMet-enriched protein, and two truncated isoforms missing either four or ten residues from the N-terminus². Complete x-ray diffraction data sets have been collected and structural analysis to beyond 2.0 Å resolution is in progress. The native and SeMet proteins crystallized in space group P6₁22 (a = b = 86 Å, c = 144 Å) and the truncated isoforms crystallized in spacegroup P2₁3(a =b=c= 87 Å); each with two molecules per asymmetric unit. The structural topology of the two monomers is similar in each spacegroup and to the NMR structure³. However, the monomer structures differ slightly from each other and the average NMR structure. These crystallographic results are the first available for the evolutionary related, but functionally diversified catalytic effector proteins from the multicomponent diiron monooxygenase family.

Portions of this research were carried out at SSRL, Stanford University and SER-CAT at APS, Argonne National Laboratory. Funded in part by the NSF (MCB-9733734) to B.G.F.

¹Pikus, J.D., Studts, J.M., Achim, C., Kauffmann, K.E., Münck, E., Steffan, R.J., McClay, K. & Fox, B.G. (1996), Biochemistry 35, 9106-19.

²Orville, A.M., Studts, J.M., Lountos, G.T., Mitchell, K.H., & Fox, B.G. (2003), Acta Cryst. D59, 572-75.

³Hemmi, H., Studts, J.M., Chae, Y.K., Song, J., Markley, J.L. & Fox, B.G.,(2001), Biochemistry 40, 3512-24.