Identification of a new class of 5 '-adenylylsulfate (APS) reductases fromsulfate-assimilating bacteria

A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Es cherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase. Th e B. cepacia gene is the most recent addition to a growing list of cysH hom ologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E. coli CysH, The evidence reported here shows that the cysH from on e of the species, Pseudomonas aeruginosa, encodes APS reductase, It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that th e enzyme is able to bypass PAPS, synthesized by the cysC product. Insertion al knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, in dicating its role in sulfate assimilation. Purified P, aeruginosa CysH expr essed as a His-tagged recombinant protein is able to reduce APS, but not PA PS, The enzyme has a specific activity of 5.8 mu mol . min(-1) . mg of prot ein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species fr om which the novel type of cysH has been cloned, indicating that this enzym e may be widespread. Although an APS reductase from dissimilatory sulfate-r educing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest tha t the dissimilatory and assimilatory APS reductases evolved convergently.