Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution

Abstract

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE(LB400)) are largely unknown. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.

Figure 1

The BPDO components and encoding genes in B. xenovorans LB400.

Figure 2

GC-MS spectra of butylboronate-derived metabolites produced (a) from 2,2'-dichlorobiphenyl and (b) from 2,6-dichlorobiphenyl by BphAE_LB400 and its variants.

Figure 3

Superposition of the catalytic center of chain AB of BphAE_LB400 (cyan) and chain AB of BphAE_LB400 bound to biphenyl (green).

Fig. 4

The 2F_obs–F_calc electron density map of chain AB of substrate-free (upper panel) and of 2,6-dichlorobiphenyl-complex (lower panel) of BphAE_p4 contoured at 1.0 σ level.

Figure 5

Superposition of active site atoms from twelve αβ dimers of BphAE_LB400 (yellow) and twelve biphenyl-bound dimers (red) with twelve αβ dimers of BphAE_p4 (green) and six dimers of 2,6-dichlorobiphenyl-bound BphAE_p4 (blue) showing the shift of the distal ring of 2,6-dichlorobiphenyl toward Gly321 and Met336.

Figure 6

(a) Superposition of active site atoms from twelve BphAE_p4 (tan) with six 2,6-dichlorobiphenyl-bound BphAE_p4 (blue) αβ dimers and (b) superposition of twelve BphAE_LB400 (yellow) with twelve biphenyl-bound BphAE_LB400 (red) dimers showing the displacement of Asp388 after substrate binding.

Figure 7

Superposition of twelve dimers of biphenyl-bound BphAE_LB400 (red) with dimers AB, CD and EF of the 2,6-dichlorobiphenyl-bound BphAE_p4 (green) showing the distances between C-2, C-3 and C-4 of 2,6-dichlorobiphenyl and the catalytic iron of dimer AB.

Figure 8

(a) Superposition of segments of dimer AB of BphAE_LB400 and of its biphenyl-bound form and (b) superposition of the corresponding segments of dimer KL of BphAE_p4and of dimer AB of its 2,6-dichlorobiphenyl-bound form. Both bound forms of BphAE_LB400 and BphAE_p4 are in yellow, native forms are in red. Dashed lines represent H-bonds of substrate complex forms (green) and of native forms (red), spheres represent water molecules.