title: Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21.
abstract: Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.
authors.affiliation: Departmento de Biología Molecular, Facultad de Veterinaria, Universidad de León, León, Spain.
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Barrientos
authors.forename: Estefanía
authors.initials: E
authors.lastname: Merino
authors.forename: Israël
authors.initials: I
authors.lastname: Casabon
authors.forename: Joaquín
authors.initials: J
authors.lastname: Rodríguez
authors.forename: Adam M
authors.initials: AM
authors.lastname: Crowe
authors.forename: Johannes
authors.initials: J
authors.lastname: Holert
authors.forename: Bodo
authors.initials: B
authors.lastname: Philipp
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
authors.forename: Elías R
authors.initials: ER
authors.lastname: Olivera
authors.forename: José M
authors.initials: JM
authors.lastname: Luengo
mesh_terms: D001647:Bile Acids and Salts / Q000378:metabolism*; D020355:Cholates / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013739:Testosterone / Q000378:metabolism
chemical_list: D001647:Bile Acids and Salts; D020355:Cholates; D013739:Testosterone; D003066:Coenzyme A Ligases