2-Hydroxycyclohexanecarboxyl Coenzyme A Dehydrogenase, an Enzyme Characteristic of the Anaerobic Benzoate Degradation Pathway Used by Rhodopseudomonas palustris

Abstract

A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD ؉ ; no activity was detected with NADP ؉ as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K m values of 10 and 200 M for 2-hydroxychc-CoA and NAD ؉ , respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate-and cyclohexanecarboxylategrown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Whereas benzoyl-CoA is reduced to a cyclohexadienecarbonyl-CoA intermediate in T. aromatica, Azoarcus spp., G. metallireducens, and S. aciditrophicus, a further reduction of the dienoyl-CoA to a monoenoyl-CoA was reported for R. palustris ( Fig. 2A) (30,31,52,104,129,142,274,279).…”

“…Despite the possibility that BCR Ta and BCR Rp may have evolved from a common ancestor, these two enzymes might differ in their biochemical properties, which could account for the single and the two successive two-electron reduction steps of benzoyl-CoA, respectively ( Fig. 2A) (30,31,52,104,128,142,274,279). The absence of korAB and korABC orthologs in the genome of R. palustris (213) suggests that the BCR electron donor-regenerating system in this phototroph is different from that characterized for denitrifying bacteria.…”

“…Although two cyclohexadienecarboxylates have been extracted from R. palustris cells grown anaerobically on benzoate, thus indicating a two-electron reduction, a cyclohex-3-ene-1-carboxylate product was detected at a 20-to 40-times-higher concentration (129). The further two-electron reduction of the initial cyclohexadienecarbonylCoA product to the monoene carbonyl-CoA, which should be energetically easy to accomplish, could be catalyzed by its own BCR Rp or by an additional enzyme that is yet unknown (30,31,129,142,274). The badF, badE, badD, and badG genes encode the ␣␤␥␦ BCR Rp heterotetramer, and these gene products, together with the badB-encoded ferredoxin, show a significant amino acid sequence identity (64 to 76%) with the corresponding bcrA, bcrB, bcrC, bcrD, and fdx gene products from T. aromatica and Magnetospirillum strains (Fig.…”

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin

“…Their approach involved reorganizing biotin biosynthetic genes into a single operon, bioBFCDA, expressed from the P tac promoter, and further optimization of this operon, by removing a stem loop after bioD and changing the RBS for bioB, which catalyzes the final step in biotin production. The chemistry of CHC degradation is well studied (Egland et al, 1997) (Kuver et al, 1995) (Pelletier and Harwood, 2000), occurring within a cluster of genes for the anaerobic degradation of benzoate and 4-hydroxybenzoate (Egland and Harwood, 2000), offering further opportunities to expand the range of substrates which can be incorporated into useful metabolites in E. coli. As the cost of DNA synthesis lowers, the ability to synthesize large pathways will not only become affordable, but will offer unique advantages over traditional cloning.…”

“…The degradation of this compound proceeds through five enzymatic steps, first creating a coenzyme A thioester, which is then metabolized via a series of β-oxidation-like reactions before a ring opening step to yield the metabolite pimeloyl-CoA ( Fig. 1) (Samanta and Harwood, 2005) (Pelletier and Harwood, 2000) (Kuver et al, 1995) (Egland et al, 1997). Pimeloyl-CoA is then further degraded enzymatically to acetyl-CoA and CO 2 (Harrison and Harwood, 2005).…”