Summary : 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 degrdn. (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 resp. 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 contg. either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ1/4-3-ketocholate and Δ1,4-3-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 deriv. Biochem. analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ1/4-3-ketocholate, and Δ1,4-3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalyzed the CoA thioesterification of HIP and its hydroxylated derivs. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degrdn. of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degrdn. of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria. [on SciFinder(R)]