Generation of Novel-Substrate-Accepting Biphenyl Dioxygenases through Segmental Random Mutagenesis and Identification of Residues Involved in Enzyme Specificity

Abstract

Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5-and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.Aryl-hydroxylating dioxygenases catalyze the addition of two hydroxy groups to vicinal carbons of their substrates, thereby destroying the aromatic system and yielding dihydrodiol compounds of cis,cis stereochemistry ( Fig. 1) (4, 6). These enzymes are of increasing interest for the degradation of aromatic pollutants (6, 10) as well as for the regio-and stereospecific functionalization of aromatic scaffolds in general (4, 16).The biphenyl dioxygenases (BphAs) are members of this family of enzymes. They have attracted attention as biocatalysts for the removal of polychlorobiphenyls. Their use in this field is hampered by the problem that pollutions typically consists of "technical" polychlorobiphenyls that are mixtures of dozens of compounds, so-called congeners. So far, only a fraction of them can be attacked by known BphAs. Therefore, enzymes with broadened and/or altered substrate ranges are required. These may be obtained either by more extensive and sophisticated screens of the natural resources (17) or by the generation of novel substrate specificities through evolution in the laboratory. In this work, we applied the latter approach.Given the persisting problem of correctly predicting the properties of designed protein variants, strategies employing random modifications are of increasing importance. Such methods of "directed evolution" have been applied before to improve substrate turnover by BphAs...

Generation by a Widely Applicable Approach of a Hybrid Dioxygenase Showing Improved Oxidation of Polychlorobiphenyls

“…First, as shown in previous publications (15,42) as well as in the present work, its substrate range frequently is narrower than that of subsequent pathway enzymes. Second, its regiospecificity of dioxygenation is a critical parameter, as it determines the (potential) site(s) of attack by the subsequent enzymes of the metabolic route (Fig.…”

Family Shuffling of Soil DNA To Change the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase

“…Altogether, the observation made regarding the catalytic features of the variant BphAEs obtained in this investigation brings to mind that changing substrate specificity is a complex process; as pointed out recently by Zielinski et al (37), it not only involves amino acid residues that interact directly with the substrate, but it also involves residues that are not part of the pocket lining and that are not predictable through structural modeling. These residues might indirectly influence the shape of the catalytic pocket or the conformation of the residues that are involved in substrate binding and positioning inside the catalytic pocket.…”

Characterization of Biphenyl Dioxygenase Sequences and Activities Encoded by the Metagenomes of Highly Polychlorobiphenyl-Contaminated Soils

“…The strains producing the prototype enzymes BphA WB15h and BphA WC18h were assayed for dioxygenation of 10 CBs that were major, minor, or not constituents of the PCB mixture found at the Wittenberg site. It had previously been shown that the BphB and BphC enzymes, which were also synthesized by the recombinant strains, were able to convert ortho,meta-dioxygenated products of all of these CBs into MCPs (33,34,43). Initial dioxygenations that formed this type of further degradable catabolites were termed "productive."…”

“…It belongs to class II of aryl-hydroxylating dioxygenases (ARHDOs) that typically hydroxylate substituted benzenes, like toluenes and biphenyls (7). This enzyme represents a catabolic bottleneck, as its substrate range is typically narrower than that of subsequent pathway enzymes (9,13,43). Moreover, its regiospecificity is a crucial parameter, as it codetermines whether the initial dioxygenation products become dead-end metabolites or can be further transformed.…”

“…A number of enzyme-engineering projects have been carried out to obtain BphAs with altered or broadened substrate ranges (6,14,19,43). Another approach is the detection and isolation of naturally occurring but so far inaccessible enzymatic activities by metagenomic methods (24,31).…”