TfdD II , one of the two chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4), cannot efficiently convert 2-chloro- cis , cis -muconate to trans -dienelactone to allow growth on 3-chlorobenzoate

Abstract

Ralstonia eutropha JMP134 (pJP4) harbors two functional gene clusters for the degradation of chlorocatechols, i.e. tfdCDEF (in short: tfd (I)) and tfdD (II) C (II) E (II) F (II) (in short: tfd (II)), which are both present on the catabolic plasmid pJP4. In this study, we compared the function of both gene clusters for degradation of chlorocatechols by constructing isolated and hybrid tfd (I)- tfd (II) clusters on plasmids in R. eutropha, by activity assays of Tfd enzymes, and by HPLC/MS of individual enzymatic catalytic steps in chlorocatechol conversion. R. eutropha containing the tfd (II) cluster alone or hybrid tfd-clusters with tfdD (II) as sole gene for chloromuconate cycloisomerase were impaired in growth on 3-chlorobenzoate, in contrast to R. eutrophaharboring the complete tfd (I) cluster. Enzyme activities for TfdD(II) and for TfdE(II) were very low in R. eutropha when induced with 3-chlorobenzoate. By contrast, a relatively high enzyme activity was found for TfdF(II). Spectral conversion assays with extracts from R. eutropha strains expressing tfdD (II) all showed accumulation of a compound with a similar UV spectrum as 2-chloro- cis,cis-muconate from 3-chlorocatechol. HPLC analysis of in vitro assays in which each individual step in 3-chlorocatechol conversion was reproduced by sequentially adding cell extracts of an Escherichia coli expressing one Tfd enzyme only demonstrated that TfdD(II) was unable to cause conversion of 2-chloro- cis,cis-muconate. No accumulation of intermediates was observed with 4-chlorocatechol. From these results, we conclude that at least TfdD(II) is a bottleneck in conversion of 3-chlorocatechol and, therefore, in efficient metabolism of 3-chlorobenzoate. This study showed the subtle functional and expression differences between similar enzymes of the tfd-encoded pathway and demonstrated that extreme care has to be taken when inferring functionality from sequence data alone.

Efficient Turnover of Chlorocatechols Is Essential for Growth of Ralstonia eutropha JMP134(pJP4) in 3-Chlorobenzoic Acid

“…2). Although differences in growth rates between the two types of derivatives may be explained in part by differences in the corresponding TfdD and TfdE activities (21,22,27,30), derivatives possessing the more active TfdC I enzyme exhibit higher growth yields and use higher concentrations of 3-CB than derivatives with the less active TfdC II enzyme. The catabolic phenotypes of the tfdC I and tfdC II mutants agree perfectly with their differential contributions to chlorocatechol turnover (Fig.…”

“…The transformation of 2-chloromuconate has been described previously as a rate-limiting step during degradation of 3-CB by the wild-type strain (28). Both TfdD I (20,41) and, more drastically, TfdD II (22,27,30) are only poorly suited to transform this substrate. Moreover, cis-dienelactone transformation is rate limiting in strains containing only tfdD II C II E II F II (22,27,30).…”

“…Both TfdD I (20,41) and, more drastically, TfdD II (22,27,30) are only poorly suited to transform this substrate. Moreover, cis-dienelactone transformation is rate limiting in strains containing only tfdD II C II E II F II (22,27,30). Theoretically, accumulation of a metabolite should produce lower growth rates and/or growth yields (as in the cases mentioned above) but not necessarily a lack of growth, as reported here for R. eutropha derivatives with single copies of the tfd modules.…”

“…A threshold copy number higher than two was determined for the clc element to allow growth of Pseudomonas putida F1 in chlorobenzene (31), and at least two copies of the tcb gene cluster are required for growth of P. putida KT2442 in 3-CB (17). In this context, it is worth mentioning that it has been speculated that the main reason for the presence of the tfdR-tfdD II C II E II F II gene cluster in pJP4 is a requirement for an active regulatory gene (tfdR) for tfd genes (21,22). However, results reported here indicate that the presence of two tfd gene modules for chlorocatechol degradation that code for the same functions in the pJP4 plasmid, a very unusual case in chloroaromatic compound degradation (38,40), might also be due to a requirement for higher TfdC activity levels.…”

Novel insights into the interplay between peripheral reactions encoded by xyl genes and the chlorocatechol pathway encoded by tfd genes for the degradation of chlorobenzoates by Ralstonia eutropha JMP134

“…The explanation for this observation is that the main muconate produced by 3-CB is 2-chloromuconate (Pieper et al, 1993). This intermediate, which is poorly metabolized by the enzyme encoded by tfdD I , is an even worse substrate for the enzyme encoded by tfdF II (Laemmli et al, 2002 ;Pe! rez-Pantoja et al, 2000, Plumeier et al, 2002.…”