The dioxygenation of various CBs by the two enzymes showed that the substrate acceptance and regiospecificity of dioxygenation by the hybrid BphA investigated in this study differed fundamentally from those of the parental enzyme. This is illustrated in Fig. .
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; (more ...)
BphA-LB400 showed major differences in the amounts of productive ortho
dioxygenation of the ortho
-, and para
-chlorinated rings. Such behavior is typical for BphAs (3
). In contrast, BphA-B4h attacked all three rings with comparable efficiencies.
Congeners with a doubly ortho
-chlorinated ring are known to generally be recalcitrant to attack by BphAs (9
). Thus, the LB400 enzyme showed no ortho
dioxygenation of 2,6-CB, although this congener possesses an unchlorinated ring, which normally is easily attacked. Similarly, the enzyme yielded only small amounts of ortho
dioxygenation products of 2,6,4′-CB. In contrast, BphA-B4h was able to catalyze significant 2′,3′-dioxygenation of both congeners.
Available data suggest that typically the 2,5-dichlorinated ring is less recalcitrant to CB dioxygenation than the 2,6-dichlorinated ring and that for some BphAs, including the enzyme of strain LB400, the 2,5-dichlorinated ring even is a preferred target (2
). Remarkably, the only sites of attack are carbons 3 and 4. Typically, 3,4-BDHDs are dead-end metabolites (12
), although a low turnover of the 3,4-DHD derived from 2,5,2′,5′-CB by BphB of Comamonas testosteroni
B-356 and by strain LB400 has been reported (6
), and our results suggest a low level of dehydrogenation and subsequent extradiol cleavage of the 3,4-DHD formed from 2,5,4′-CB. Moreover, transformation of chlorinated meta
-BDHDs by BphB and BphC may form acylchlorides, which may inactivate enzymes by attack of side chains in the active site. In contrast, ring cleavage of a chlorinated ortho
-DHB between carbons 1 and 2 or 1 and 6, respectively, can never yield an acylchloride. Contrary to the LB400 enzyme, BphA-B4h converted 2,5,4′-CB into only marginal amounts of the 3,4-dioxygenated metabolite but into almost 100-fold-larger amounts of the BDHD resulting from the more favorable ortho
dioxygenation of the other ring.
In summary, BphA-B4h showed a higher regiospecificity of dioxygenation. In only one case did the main product represent less than 95% of the dioxygenated CB (Table ). BphA-B4h also possessed a greater preference for ortho,meta dioxygenation. Thus, BphA-LB400 formed meta,para-dioxygenated products from five of the six substrates (Fig. ), with an average contribution of 61% (Table ), while BphA-B4h yielded such metabolites only from three CBs, with a major contribution only in the case of 2,6-CB (Table ).
Interestingly, the subsequent enzymes of the bph
-encoded metabolic pathway of strain LB400 were able to transform most of those metabolites produced by the hybrid BphA, with which they normally are not confronted. Thus, 2′,3′-dioxygenated 2,6-CB was converted at least into the respective HOPDA, and 5,6-dioxygenated 2,2′-CB was transformed to 2-CBA. These results show that subsequent metabolic enzymes can possess a broader substrate spectrum than the initial pathway enzyme. This is particularly remarkable for the hydrolase BphD, which has been reported to constitute a major bottleneck (14
On the other hand, it is directly apparent from a correlation of Tables and that the products of 2,3 dioxygenation of 4,4′-CB and of 2′,3′-dioxygenation of 2,5,4′-, 2,6-, and 2,6,4′-CB were not or only marginally converted into the respective CBAs by the subsequent pathway enzymes. When 4,4′- and 2,5,4′-CB were dioxygenated by BphA-B4h, significant amounts of the meta-fission products were found. This indicates that these HOPDAs were not or were very slowly converted by the hydrolase BphD (Fig. ). Only small or no detectable quantities of HOPDAs were formed from the BDHDs generated by 2′,3′ dioxygenation of 2,6- and 2,6,4′-CB. This indicates a problem with the dehydrogenase BphB and/or the extradiol dioxygenase BphC (Fig. ). A comparison of the structures of the respective ortho,meta-dioxygenated BDHDs suggests that it is the double ortho substitution of the nonoxidized ring which prevents efficient turnover by one or both of these enzymes.
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. (more ...)
We note that Seah et al. (32
) found that HOPDA chlorinated at position 3 was hydrolyzed by BphD-LB400 at a 500-fold lower rate than the unchlorinated metabolite. This is consistent with no or only marginal conversions of the 3,10-dichlorinated or 3,8,11-trichlorinated HOPDA, resulting from 4,4′- or 2,5,4′-CB, respectively, into 4-CBA (Fig. ). The same authors also reported that HOPDA chlorinated only at position 4 was hydrolytically cleaved at a 10,000-fold-lower rate than the unsubstituted compound (32
). Thus, the observed conversion of 3,3′-CB into a 4,9-dichlorinated HOPDA that was further metabolized to 3-CBA suggests that, unexpectedly, the additional chlorine substituent at the nonoxidized ring significantly enhances this rate. A positive electronic effect appears unlikely, as recently, Speare et al. (37
) have shown that electron-withdrawing substituents at the nonoxidized ring decrease the rate of HOPDA hydrolysis by BphD-LB400.
The exchange of the BphA1 core segment resulted in 24 amino acid differences between the LB400 and the hybrid sequence (see the supplemental material). Therefore, alterations in catalytic behavior cannot directly be ascribed to single or a few amino acid substitutions. However, several of the replaced residues have previously been exchanged, either singly or in groups of two and three (22
). In most cases, this did not result in significant changes of the examined properties. An exception was the region comprising amino acids 335 to 341 (LB400 numbering), where four of the present substitutions are located. However, more than these exchanges are likely to play a role, because seemingly “unimportant” residues may exert significant effects when replaced in concert with additional amino acids. Such a context dependence of substitutions has repeatedly been reported (22
The fundamental differences in substrate and product spectra between the parent and hybrid enzymes demonstrate that BphAs with novel catabolic potential can be obtained through a rapid approach involving PCR amplification of partial genes encoding the large subunit of aryl-hydroxylating dioxygenases and their fusion with cloned “helper” genes and gene segments to reconstitute a complete aryl-hydroxylating dioxygenase system. The speed of this approach and the possibility of carrying out PCR amplifications directly with metagenomic DNA samples may permit rapid access to a much larger part of natural BphA diversity than was previously possible. Such an approach could thus favorably complement or supplement methods of artificial evolution (14
) for the acquisition of novel dioxygenase activities.