The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

Abstract

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C 5 acyl side chain of the C 24 steroid compound cholate involves the C 22 intermediate 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C 24 steroid (22E)-7␣,12␣-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7␣,12␣,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A-and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD ؉ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.

Pathogen roid rage: Cholesterol utilization byMycobacterium tuberculosis

“…This activity is required for salvaging cholesterol-24-oic acids that are formed as a result of CoA ester hydrolysis during β-oxidation or that are formed through an alternate pathway ( vide infra ) (Holert et al, 2013a). …”

“…strain Chol1, it has been proposed on the basis of gene knockout and metabolite studies, and assays in cell lysates that cleavage of the 24-oyl-CoA intermediate produces an aldehyde metabolite rather than a thioester via a classical β-oxidation pathway. This cleavage is catalyzed by an aldolase (shy) that is homolgous to Ltp3 from R. rhodochrous DSM 43269 (Holert et al, 2013a, Holert et al, 2013b). The aldehyde is then oxidized to the carboxylic acid by an aldehyde dehydrogenase (sad) and esterified by a CoA ligase.…”

“…The individual steps in the classical β-oxidation pathway are shown for the second cycle with solid arrows. Recently proposed alternate steps via an aldehyde (Holert et al, 2013a, Holert et al, 2013b) are shown as dashed arrows. The individual steps catalyzed in the third cycle are shown in Figure 5.…”

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

“…CasI likely functions in reassimilation of the metabolites which accumulate when RHA1 grows on cholate (27). CasI orthologs likely function in further degradation of an intermediate resulting from the above-mentioned aldolytic cleavage of the cholate side chain by Proteobacteria (12,24). It is unclear whether such aldolytic cleavage occurs in Actinobacteria.…”

“…Holert et al recently showed evidence for degradation of the cholate side chain in Proteobacteria via a variation of ␤-oxidation, in which an acetyl-CoA moiety is removed by aldolytic, rather than thiolytic, cleavage (12,24). The resulting aldehyde is proposed to be oxidized to an acid and reactivated by CoA thioesterification for another round of ␤-oxidation.…”