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Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH-
and O(2)-dependent hydroxylation of toluene to form p-cresol. The complex
consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a
diiron hydroxylase [T4moH, with (alphabetagamma)(2) quaternary structure], and a
catalytic effector protein (T4moD). The solution structure of the 102-amino acid
T4moD effector protein has been determined from 2D and 3D (1)H, (13)C, and (15)N
NMR spectroscopic data. The structural model was refined through simulated
annealing by molecular dynamics in torsion angle space (DYANA software) with
input from 1467 experimental constraints, comprising 1259 distance constraints
obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80
hydrogen bond constraints. Of 60 conformers that met the acceptance criteria,
the 20 that best satisfied the input constraints were selected to represent the
solution structure. With exclusion of the ill-defined N- and C-terminal segments
(Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20
conformers with respect to the mean coordinates was 0.71 A for the backbone and
1.24 A for all non-hydrogen atoms. The secondary structure of T4moD consists of
three alpha-helices and seven beta-strands arranged in an N-terminal
betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology.
Although the published NMR structures of the methane monooxygenase effector
proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus
(Bath) have a similar secondary structure topology, their three-dimensional
structures differ from that of T4moD. The major differences in the structures of
the three effector proteins are in the relative orientations of the two
beta-sheets and the interactions between the alpha-helices in the two domains.
The structure of T4moD is closer to that of the methane monooxygenase effector
protein from M. capsulatus (Bath) than that from M. trichosporium OB3b. The
specificity of T4moD as an effector protein was investigated by replacing it in
reconstituted T4MO complexes with effector proteins from monooxygenases from
other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene
3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and
Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase). The results showed that
the closely related TbuV effector protein (55% sequence identity) provided
partial activation of the complex, whereas the more distantly related TbmC (34%
sequence identity) and S1 (29% sequence identity) did not. The (1)H NMR chemical
shifts of the side-chain amide protons of Asn34, a conserved, structurally
relevant amino acid, were found to be similar in spectra of effector proteins
T4moD and TbuV but not in the spectrum of TbmC. This suggests that the region
around Asn34 may be involved in structural aspects contributing to functional
specificity.
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