Steady-state Kinetic Characterization and Crystallization of a Polychlorinated Biphenyl-transforming Dioxygenase

Abstract

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe 2 S 2 center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 Å. Steady-state kinetics assays demonstrated that BPDO had an apparent k cat /K m for biphenyl of (1. The microbial catabolic activities responsible for the degradation of aromatic compounds constitute an essential link in the global carbon cycle. These activities are of considerable practical interest due to their potential to destroy toxic, persistent pollutants, a strategy known as bioremediation (1). In the case of highly chlorinated, structurally diverse xenobiotics such as PCBs, 1 the development of a practical bioremediation technology has been limited in part by the failure of existing microbial catabolic activities to effectively degrade these compounds (1, 2). This failure may arise because these activities have not yet evolved to degrade compounds that have only recently been introduced into the biosphere. An important aspect of the adaptation of catabolic activities for bioremediation is the study of the structure and function of key catabolic enzymes. Such studies provide insight into the molecular basis of an important biological process, thereby facilitating the modification of enzyme specificity and the design of novel metabolic pathways.2BPDO catalyzes the initial reaction in the aerobic degradation of biphenyl and some PCBs. BPDO is a typical aromatic ring-hydroxylating dioxygenase, utilizing O 2 and electrons originating from NADH to transform biphenyl to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene ( Fig. 1) (3, 4). This dihydroxylation prepares the ring for subsequent degradation by ring cleavage enzymes. The enzyme comprises an FAD-containing reductase (BphG), a Rieske-type ferredoxin (BphF), and a two-subunit oxygenase of ␣ 3 ␤ 3 constitution that contains a Rieske-type Fe 2 S 2 cluster and a mononuclear iron center. Accordingly, BPDO has been classified as a group IIB aromatic ring-hydroxylating dioxygenase together with benzene and toluene dioxygenases (5). Structural and spectroscopic studies of related dioxygenases indicate that the mononuclear iron center orchestrates substrate transformation (reviewed in Ref. 6). BphG, BphF, and the oxygenase Fe 2 S 2 cluster function to transfer electrons from NADH to this center.BPDO is a major determinant of the PCB-catabolizing capabilities of biphenyl-degrading strains, and the enzymes from different strains possess significantly different congener-transforming abilities. For example, BPDO LB400 from Burkholderia cepacia LB400 transforms a much broader range of congeners than BPDO KF707 from Pseudomonas ps...

Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

“…3). These spectra are similar to those of the oxygenases of other ring-hydroxylating dioxygenases (17,29). The extinction coefficient of ISP TADO was 83.4 mM Ϫ1 cm Ϫ1 at 323 nm (25 mM HEPES, pH 7.3; 25°C) based on its sulfur content.…”

“…Production of TADO components in suitably engineered pseudomonad strains greatly increased the amount of expressed, soluble protein, as was observed for biphenyl dioxygenase (BPDO [29]). Thus, the yield of ca.…”

Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III

“…Bedard et al (26) have previously reported the fact that the more recalcitrant PCB congeners are degraded more efficiently when present at very low concentration. There is no clear explanation for this observation; however, substrate inhibition of BPDO reaction caused by uncoupling of NADH oxidation to substrate oxidation has been demonstrated for several chlorobiphenyls (27). Nevertheless, clearly, variant p4 has acquired the capacity to oxygenate this co-planar congener.…”

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls(PCBs).

“…The ferredoxin and ferredoxin reductase act as a short electron transport system from NADH to reduce the terminal dioxygenase (Broadus and Haddock, 1998). BphA1 and BphA2 are associated as an a3b3 heterohexamer Hirose et al, 1994b;Imbeault et al, 2000). BphA1 contains the motif Cys-X-His-X17-Cys-X2-His that forms a Rieske-type [2Fe-2S] cluster.…”

“…An analysis of the substrate specificity pattern of these chimeras toward selected PCB congeners indicate that the catalytic capacity of hybrid enzymes comprised of an a and a b subunit recruited from distinct BP Dox is not determined specifically by either of the two subunits. Imbeault et al (2000), crystallized an oxygenase component of BP Dox of Comamonas testosteroni B-356 and got preliminary x-ray differentiation data.…”