Generation by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls

Abstract

Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology 149:1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the bphA gene cluster of Burkholderia xenovorans LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from Pseudomonas sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at meta and para carbons. It attacked ortho-, meta-, and para-chlorinated rings with comparable efficiencies. It gave significantly higher yields in ortho,meta-dioxygenation of recalcitrant congeners containing a doubly ortho-chlorinated ring. While the parental enzyme yielded mainly unproductive meta, para dioxygenation of 2,5,4'-CB, the hybrid predominantly converted this congener into an ortho,meta-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.

FIG. 1.

Upper pathway for catabolism of biphenyls encoded by the bph locus of B. xenovorans LB400. Compounds: 1, biphenyl; 2, biphenyl-2,3-dihydro-2,3-diol (BDHD); 3, 2,3-dihydroxybiphenyl (DHB); 4, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA); 5a, 2-hydroxypenta-2,4-dienoic acid; 5b, benzoic acid. Enzymes: BphA, biphenyl 2,3-dioxygenase; BphB, 2,3-dihydro-2,3-dihydroxybiphenyl 2,3-dehydrogenase; BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase.

FIG. 2.

Generation of the fusion dioxygenase. The top line shows a representation of the bphA gene cluster of B. xenovorans LB400, encoding alpha and beta subunits (bphA1/bphA2), ferredoxin (bphA3), and ferredoxin reductase (bphA4). The hatched bphA1 segment between the restriction sites was exchanged with a PCR amplicon. The bottom line shows a representation of the alpha subunit. Catalytic and Rieske domains are shown in gray or are hatched, respectively. Horizontally connected vertical bars indicate sites encoding amino acid ligands of the Rieske iron-sulfur cluster ([2Fe-2S]) and of the active site mononuclear iron (mono-Fe). The part encoded by the amplicon and the flanking amino acid positions of the recipient subunit are indicated.

FIG. 3.

Time course of HOPDA formation. Absorbance (milliunits) at absorption maxima (mA_max) are shown. The substrates were 3,3′-CB (circles) and 2,5,4′-CB (squares). Both substrates were initially oxidized by BphA-B4h.

FIG. 4.

Overview of the regiospecificity of CB dioxygenation by BphA-B4h and -LB400. Regiospecificities of attack are symbolized by O₂ molecules with arrows. Relative quantities are indicated as follows: black arrows, >33% of total dioxygenation; gray arrows, 10 to 33% of total dioxygenation; light gray arrows, <10% of total dioxygenation. For experimental evidence of site assignments and further details, see the text and Table 1. A lack of experimental evidence for the assignment of an oxidation site is indicated by “S?”; a lack of experimental evidence for the assignment of the relative quantity of oxidation is indicated by “Q?”

FIG. 5.

Bottlenecks in the metabolism of specific ortho,meta-dioxygenated chlorinated BDHDs by enzymes BphB, BphC, and BphD of the pathway from B. xenovorans LB400. Thin arrows symbolize low transformation; crossed-out thin arrows symbolize no detectable transformation.