4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases

Abstract

The anaerobic degradation of 4-hydroxybenzoate is initiated by the formation of 4-hydroxybenzoyl coenzyme A, with the next step proposed to be a dehydroxylation to benzoyl coenzyme A, the starting compound for a central pathway of aromatic compound ring reduction and cleavage. Three open reading frames, divergently transcribed from the 4-hydroxybenzoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris. These genes, named hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respectively. The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nicotine metabolism that have conserved binding sites for [2Fe-2S] clusters and a molybdenum cofactor. Cassette disruption of the hbaB gene yielded a mutant that was unable to grow anaerobically on 4-hydroxybenzoate but grew normally on benzoate. The hbaB mutant cells did not accumulate [ 14 C]benzoyl coenzyme A during short-term uptake of [ 14 C]4-hydroxybenzoate, but benzoyl coenzyme A was the major radioactive metabolite formed by the wild type. In addition, crude extracts of the mutant failed to convert 4-hydroxybenzoyl coenzyme A to benzoyl coenzyme A. This evidence indicates that the hbaBCD genes encode the subunits of a 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating). The sizes of the specified polypeptides are similar to those reported for 4-hydroxybenzoyl coenzyme A reductase isolated from the denitrifying bacterium Thauera aromatica. The amino acid consensus sequence for a molybdenum cofactor binding site is in HbaC. This cofactor appears to be an essential component because anaerobic growth of R. palustris on 4-hydroxybenzoate, but not on benzoate, was retarded unless 0.1 M molybdate was added to the medium. Neither tungstate nor vanadate replaced molybdate, and tungstate competitively inhibited growth stimulation by molybdate.

Shedding light on anaerobic benzene ring degradation: a process unique to prokaryotes?

“…The deduced amino acid sequences of the HbaBCD polypeptides show significant similarities (25 to 30% amino acid identities) to a group of molybdenumcontaining iron-sulfur proteins that are involved in aerobic CO oxidation and xanthine and nicotine hydroxylation. Consistent with this is the observation that R. palustris cultures require added molybdate for optimal growth on 4-OHBen, but not for growth on benzoate (29).…”

“…The genes that encode the three subunits of the 4-OHbenzoyl-CoA reductase from R. palustris are adjacent to and divergently transcribed from the gene encoding the 4-OHBenCoA ligase (29). Disruption of the first gene in the three-gene (hbaBCD) sequence with an antibiotic resistance cassette resulted in the generation of a mutant that was unable to grow on 4-OHBen and that lacked the ability to dehydroxylate 4-OHbenzoyl-CoA to benzoyl-CoA.…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Consequently, BCR catalysis requires the input of external energy (ATP) to promote electron transfer at a physiological rate, whereas 4-hydroxybenzoyl-CoA reductase does not (31). The hbaBCD genes from R. palustris have been cloned and expressed in E. coli cells, but no 4-hydroxybenzoyl-CoA was detected, suggesting that the active enzyme is not easily reconstituted from separately synthesized subunits and/or that some of the enzyme cofactors, such as molybdopterin cytosine dinucleotide, are not synthesized in E. coli (127). In both T. aromatica and R. palustris, the genes encoding the 4-hydroxybenzoyl-CoA reductase are associated with genes encoding a putative transcriptional regulator of the MarR family (orf1) and a transcriptional activator of the fumarate nitrate reductase (FNR)/cyclic AMP receptor protein (CRP) superfamily (hbaR), respectively ( Fig.…”

“…The amino acid sequences of the three subunits of the T. aromatica (HcrCAB) and R. palustris (HbaBCD) 4-hydroxybenzoyl-CoA reductases showed 47 to 62% identity (40,127). Interestingly, among all Mo-containing members of the xanthine oxidase family (172), the two enzymes contain a unique extra sequence domain in the HcrB (HbaD) subunit coordinating the [4Fe-4S] cluster, which plays an essential role in mediating two-electron transfer from the low-potential donor, reduced ferredoxin to the other redox centers.…”

Benzoyl Coenzyme A Pathway-Mediated Metabolism of meta -Hydroxy-Aromatic Acids in Rhodopseudomonas palustris

“…In both organisms, specific CoA-dependent ligases catalyze the ATP-dependent synthesis of pHB-CoA (HbaA, EC 6.2.1.27) (9-11) and benzoylCoA (BadA, EC 6.2.1.25) (12)(13)(14). The pHB-CoA thioester is subsequently dehydroxylated by pHB-CoA reductase (HbaBCD, EC 1.3.7.9) to produce benzoyl-CoA (15)(16)(17). Benzoyl-CoA dearomatization is catalyzed by benzoyl-CoA reductase (BadDEFG, EC 1.3.7.8) forming aliphatic intermediates (1,5-diene-cyclohexanoyl-CoA in T. aromatica [18,19] and 1-ene-cyclohexanoylCoA in R. palustris [20]) that serve as carbon sources and reducing power in catabolic pathways that ultimately yield acetyl-CoA, CO 2 , and NADH (21)(22)(23)(24).…”