Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida

Abstract

Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, alpha and beta; the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the alpha subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase alpha subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the alpha subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.

FIG. 1.

(A) Diagram of the genes encoding ISP_BEDα, ISP_TODα, ISP_BODα, ISP_TEDα, ISP_BOD2α, and ISP_BOD3α and relative activities of the corresponding cell extracts. (B) Diagram of the single and multiple mutations created in the ISP_BEDα C-terminal region and the effects of the mutations on the relative activity. The bars indicate the open reading frames encoding ISP α subunit proteins. The nonconserved amino acids targeted for mutation are indicated by solid boxes. The letters above the bars indicate translated amino acids in ISP_BEDα, and the letters below the bars indicate amino acids in ISP_TODα. The numbering corresponds to the numbering for the amino acids in the ISP_BEDα sequence. Solid diamonds indicate the conserved histidine (H98 and H119) and cysteine (C96 and C116) residues responsible for coordination of the [2Fe-2S] cluster. Solid circles indicate the conserved amino acids (two histidines [H222 and H228] and one aspartate [D376]) involved in coordination of the nonheme iron. The EcoRI, KpnI, BstEII, SnaBI, MluI, and HindIII sites are the restriction sites used for cloning the recombinant α subunit genes. The relative activities with toluene (tol) and ethylbenzene (ethylben) are compared to the activity with benzene (ben), and the data are based on the rate of O₂ uptake measured polarographically by using ISP_BED or ISP_TOD β subunits, as described in Materials and Methods (means of three determinations).

FIG. 2.

SDS-PAGE-resolved E. coli cell extracts expressing recombinant ISP α subunits. The numbers in parentheses indicate the levels of expression of the soluble ISP α subunit in the cell extract (in milligrams per milligram of total protein; means of two determinations). (A) Wild-type and chimeric ISP α-subunit cell extracts. Lane 1, purified ISP_BEDα (2 μg); lanes 2 to 7, cell extracts (5 μg of protein per lane) from E. coli strains containing plasmids pJRM504 (ISP_BEDα), pJRM5041 (ISP_BODα), pJRM5042 (ISP_TODα), pJRM5043 (ISP_TEDα), pJRM5041-2 (ISP_BOD3α), and pJRM5041-1 (ISP_BOD2α), respectively. (B) Wild-type and mutant ISP α subunit cell extracts. Lanes 1 to 12, cell extracts (5 μg of protein per lane) from E. coli strains containing plasmids pKK223-3, pJRM504 (ISP_BEDα), pJRM504M1 [ISP_BEDα(L285W)], pJRM504M2 [ISP_BEDα(A291S)], pJRM504M7 [ISP_BEDα(G404D)], pJRM504M3 [ISP_BEDα(L285W/A291S)], pJRM504M6 [ISP_BEDα(L285W/A291S/G404D)], pJRM504M4 [ISP_BEDα(I301V/T305S/I307L/L309V)], pJRM504M5 [ISP_BEDα(V324I/I327V)], pJRM504M8 [ISP_BEDα(I412V)], pJRM504M9 [ISP_BEDα(K436R)], and pJRM504M10 [ISP_BEDα(E444D)], respectively.

FIG. 3.

Autoradiograms of the products formed by using [¹⁴C]benzene (A), [¹⁴C]toluene (B), and [¹⁴C]ethylbenzene (C) by wild-type, chimeric, and mutant ISP_BEDα subunits combined with the ISP_BEDβ subunit and coexpressed ISP_BEDα-ISP_BEDβ, ISP_TODα-ISP_BEDβ, and ISP_BODα-ISP_BEDβ in the dioxygenase assay. The R_f values of the radiolabeled products are indicated on the left. The numbers on the autoradiograms indicate the percentages of the chain monohydroxylated and cis-diol products. The values are averages of two determinations. A repeat experiment gave similar results. Details of the experiment are described in Materials and Methods.

FIG. 4.

Structural representation of the putative substrate entry channel in ISP_BEDα(I301V/T305S/I307L/L309V). The protein is represented in spacefill. Iron is indicated by red, and the four amino acid substitutions (I301V, T305S, I307L, and L309V) are indicated by blue spheres. The view is a section through the substrate channel, and the arrow indicates the putative route that the substrate takes to get to the iron catalytic site. The model was generated with the SWISS-MODEL program (; http://expasy.hcuge.ch/swissmod/SWISS-MODEL.html) and was displayed by using RasWin Molecular Graphics Window, version 2.6 (43).

FIG. 5.

Alternative pathways for dioxygenase and monooxygenase activities and uncoupling. Dihydroxylation results from the addition of O₂ and two-electron reduction to the peroxo derivative. In the structure of naphthalene dioxygenase with O₂ and substrate in the active site (24), the oxygen is bound side-on to the iron and next to the aromatic ring. Addition of both oxygen atoms, with two protons, would lead to addition of two —OH groups. The monooxygenation reactions are concerted reactions in which one oxygen atom is protonated and reduced to H₂O and the other oxygen atom is inserted into the C—H bond of the side chain. The uncoupling reaction is shown as a protonation of the Fe-peroxo species, releasing H₂O₂.