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 (RO) that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme’s oxygenase component (BphAELB400) are largely unknown. BphAEp4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAELB400 yet differs from the parent oxygenase at only two positions: T335A/F336M. Herein, we compare the structure of BphAELB400 and BphAEp4 and examine the biochemical properties of two BphAELB400 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 did not contact biphenyl, but contributed significantly to expansion of the enzyme’s substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and non-bonded 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, increasing the space available to accommodate the doubly-ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how ROs can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.

Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase

“…Interpretable electron density was not observed for residues 144 to 152 of the ␣-subunit. These residues were also absent in the wild-type BphAE LB400 structure (11). Similar to other Rieske-type dioxygenases, each ␣-subunit binds a [2Fe-2S] Rieske-type cluster and a mononuclear Fe 2ϩ ion at the active site, which is coordinated to two His residues (His233 and His239), an Asp residue (Asp388), and one or more water molecules.…”

“…DDT (99% pure) was obtained from Sigma-Aldrich, and 2,3,4=-trichlorobiphenyl and 2,2=,5,5=-tetrachlorobiphenyl were obtained from Ultra Scientific. The ability of BphAE LB400 , BphAE B356 , and BphAE II9 to metabolize 2,3,4=-trichlorobiphenyl, 2,2=,5,5=-tetrachlorobiphenyl, and DDT was assessed by using BPDO systems reconstituted from His-tagged components produced and purified as described previously (11). The enzyme assays were performed with 200 l of a solution containing 100 to 800 nmol the substrate and buffered with 50 mM MES (pH 6.0) at 37°C as described previously (35).…”

“…In liquid culture depletion assays, this variant eliminates several congeners better than does BphAE LB400 , including the very persistent 2,6-dichlorobiphenyl (25). Structural analysis revealed that the altered substrate specificity of BphAE P4 is associated with both changes in direct side-chain-substrate interactions and changes in residue-residue interactions near the active site; the latter appear to relieve constraints on the induced fit between the enzyme and substrates (11).…”

“…Bacteria can utilize the Bph pathway to cometabolize a variety of PCB congeners with differential effectiveness, and different PCB congeners are transformed in a straindependent fashion (10). Until now, structural studies have been done for all four enzymes to understand the detailed mechanism that underlies this pathway (11)(12)(13)(14)(15)(16).…”

“…As is typical of ring-hydroxylating ROs, the oxygenase is a 3-fold symmetric heterohexamer assembled from three larger BphA (␣) subunits and three smaller BphE (␤) subunits such that the overall shape resembles that of a mushroom, with the three ␣-subunits forming the cap and the three ␤-subunits forming the stem. Each ␣-sub-unit includes a [2Fe-2S] Rieske-type cluster (His 2 Cys 2 ligation) involved in electron transfer from external reductants to an active site containing a mononuclear Fe 2ϩ ion coordinated by two His residues, an Asp residue (His233, His239, and Asp388 in BphAE LB400 [11]), and a conserved water molecule. The 3-fold symmetric arrangement of ␣␤-dimers brings the [2Fe-2S] cluster of one dimer close to the active site of another, such that the Rieske center of one ␣-subunit is 12 Å from the Fe 2ϩ atom of the neighboring subunit.…”