Functional analyses of three acyl‐ CoA synthetases involved in bile acid degradation in P seudomonas putida DOC 21

Summary P seudomonas putida   DOC 21, 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 Δ stdA1 and Δ stdA2 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 Δ stdA3 mutant grew poorly in media containing either testosterone or AD . When cells were grown with succinate in the presence of cholate, Δ stdA1 accumulated Δ 1/4 ‐3‐ketocholate and Δ 1,4 ‐3‐ketocholate, whereas Δ stdA2 only accumulated 7α,12α‐dihydroxy‐3‐oxopregna‐1,4‐diene‐20‐carboxylate ( DHOPDC ). When incubated with testosterone or bile acids, Δ stdA3 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 StdA 2 transformed DHOPDC to DHOPDC ‐ CoA . In contrast, purified StdA 3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA 1, StdA 2 and StdA 3 are acyl‐ CoA synthetases required for the complete degradation of bile acids: StdA 1 and StdA 2 are involved in degrading the C ‐17 acyl chain, whereas StdA 3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between P roteobacteria and A ctinobacteria .