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. 2000 May;182(10):2753-60.
doi: 10.1128/jb.182.10.2753-2760.2000.

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

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

D A Pelletier et al. J Bacteriol. .
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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 microM 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 cyclohexanecarboxylate-grown 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.

Figures

FIG. 1
FIG. 1
Comparison of anaerobic benzoate degradation by R. palustris and T. aromatica. Reactions involved in funneling cyclohexanecarboxylate into the anaerobic benzoate degradation pathway in R. palustris are shown. Solid arrows indicate enzymatic activities that have been purified from either R. palustris or T. aromatica (1, 6, 11, 23, 25, 26, 33). Dashed arrows indicate hypothetical enzymatic reactions. Assignment of gene products from R. palustris (Fig. 2) and T. aromatica (7) to specific steps is indicated. redBadB, reduced BadB; redFdx, reduced ferredoxin.
FIG. 2
FIG. 2
(A) Map of bad (benzoic acid degradation) gene cluster from R. palustris. Arrows indicate transcriptional units. The genes encode enzymes of anaerobic benzoate or cyclohexanecarboxylate degradation, as indicated in Fig. 1. (B) Nucleotide sequence of the badH promoter region, with numbering indicating the start site of badH transcription (+1) and the −10 and −35 (underlined) nucleotides. (C) Mapping of the badH transcriptional start site by primer extension. The position of the primer extension product is indicated by the arrow (lane PE). RNA for the primer extension reaction was isolated from benzoate-grown cells. A sequence ladder generated with the same primer is shown.
FIG. 3
FIG. 3
Amino acid sequence alignment of BadH with selected members of the SDR family. The abbreviation, enzyme, number of amino acids, organism, and accession number, respectively, for each family member follow: BadH, 2-hydroxycyclohexanecarboxyl-CoA dehydrogenase, 255, R. palustris, and U75363 (10); PhbB, acetoacetyl-CoA reductase, 241, Rhizobium meliloti, and P50205 (43); FabG, 3-ketoacyl-acyl carrier protein reductase, 244, E. coli, and P25716 (36); NodG, nodulation protein G, 246, Azospirillum brasilense, and P17611 (44); 20HSD, 20β-hydroxysteroid dehydrogenase, 255, S. exfoliatus, and P19992 (28); and 7αHSD, 7α-hydroxysteroid dehydrogenase, 255, E. coli, and P25529 (46). Alignment was created using CLUSTALW (version 1.74), and shading was done with the program BOXSHADE. Black shading indicates 100% amino acid identity, and gray shading indicates 100% similarity. ∗, active-site amino acids. The boxed sequence is the proposed pyridine nucleotide-binding site, and the solid black line denotes the SDR family signature (20).
FIG. 4
FIG. 4
SDS-PAGE analysis of active protein fractions obtained during purification of His-BadH. Lanes: CE, crude cell extract (20 μg); Ni-C, nickel column pooled fractions (1 μg); DS-C, Hitrap desalting column pooled fractions (1 μg); MW, protein ladder (Bio-Rad). Numbers on the right are molecular weights, in thousands.
FIG. 5
FIG. 5
Time course of 2-hydroxychc-CoA dehydrogenase activity. Activity was measured spectrophotometrically by monitoring the reduction of NAD+ at 340 nm using standard assay conditions as described in Materials and Methods. Reaction mixtures contained purified His-BadH (0.8 μg) plus purified 2-ketocyclohexanecarboxyl-CoA hydrolase (10 μg) (triangles), hydrazine (60 mM) (squares), or neither hydrazine nor 2-ketocyclohexanecarboxyl-CoA hydrolase (circles).
FIG. 6
FIG. 6
Time course of anaerobic benzoate transformation by cultures of CGA702 (badH::Kmr). Cells were grown anaerobically in mineral medium containing 10 mM succinate and 1.5 mM benzoate. Aliquots were removed at various time points, cells were removed by centrifugation, and supernatants were analyzed by C18 reverse-phase HPLC for the presence of benzoate (squares) and cyclohex-1-ene-carboxylate (circles). Growth (represented by triangles) was monitored spectrophotometrically by monitoring optical density at 660 nm (OD660 nm). Data points are averages of duplicates.

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