4-HYDROXYBENZOYL COENZYME-A REDUCTASE (DEHYDROXYLATING) IS REQUIRED FOR ANAEROBIC DEGRADATION OF 4-HYDROXYBENZOATE BY RHODOPSEUDOMONAS-PALUSTRIS AND SHARES FEATURES WITH MOLYBDENUM-CONTAINING HYDROXYLASES

The anaerobic degradation of 4-hydroxybenzoate is initiated by the for mation 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. Th ree open reading frames, divergently transcribed from the 4-hydroxyben zoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris, These genes, n amed hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respect ively, The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nic otine metabolism that have conserved binding sites for [2Fe-2S] cluste rs and a molybdenum cofactor, Cassette disruption of the hbaB gene yie lded a mutant that was unable to grow anaerobically on 4-hydroxybenzoa te but grew normally on benzoate, The hbaB mutant cells did not accumu late [C-14] benzoyl coenzyme A during short-term uptake of [C-14] 4-hy droxybenzoate, but benzoyl coenzyme A was the major radioactive metabo lite formed by the wild type, In addition, crude extracts of the mutan t 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 o f the specified polypeptides are similar to those reported for 4-hydro xybenzoyl coenzyme A reductase isolated from the denitrifying bacteriu m Thauera aromatica. The amino acid consensus sequence for a molybdenu m cofactor binding site is in HbaC, This cofactor appears to be an ess ential component because anaerobic growth of R. palustris on 4-hydroxy benzoate, but not on benzoate, was retarded unless 0.1 mu M molybdate was added to the medium, Neither tungstate nor vanadate replaced molyb date, and tungstate competitively inhibited growth stimulation by moly bdate.