IpdE1-IpdE2 Is a Heterotetrameric Acyl Coenzyme A Dehydrogenase That Is Widely Distributed in Steroid-Degrading Bacteria

Abstract

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2β2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α­(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and β-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α­(3′-prop-1-enoate)-7aβ-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via β-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2β2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.

Degradation of Bile Acids by Soil and Water Bacteria

“…In bile acid degradation, this reaction sequence can depend on the organism and on the hydroxylation pattern of the bile acid substrate, which is discussed in more detail below. In general, the side chain of HIP-CoA is dehydrogenated by heterotetrameric ACADs similar to the ones involved in steroid side-chain degradation [ 126 ]. In P. stutzeri Chol1 and C. testosteroni TA441, these ACADs are encoded by the genes scd3A / scd3B and scdC1/scdC2 , respectively [ 84 , 127 ].…”

A Key Glycine in Bacterial Steroid-Degrading Acyl-CoA Dehydrogenases Allows Flavin-Ring Repositioning and Modulates Substrate Side Chain Specificity

“…+++ indicates only specific activity has been determined for this enzyme (41 μM min −1 mg −1 ). 26 different conditions, with unique packing interactions, and with the flavin in different apparent redox states (the ring in the Gly363Ala structure is bent at an angle of ∼23°, implying the reduced, FADH 2 form; experiments on the close homologue FadE26-FadE27 have established that X-ray radiation can reduce the cofactor in these enzymes during data collection 7 ). However, shifts of this magnitude do not typically occur in ACAD enzymes, and our interpretation is that these differences arise primarily as a consequence of the Gly363Ala variation that preferentially stabilizes a naturally occurring structural state of the enzyme, thereby enabling new crystallization conditions that stabilize a distinct crystal packing arrangement.…”

“…In FadE34 and CasC, the two-domain ACADs that are specific to five-carbon side chain steroid substrates in the cholesterol and bile acid degradation pathways of M. tuberculosis and R. jostii RHA1, respectively, this residue is replaced by alanine. Finally, in M. tuberculosis FadE30 (also known as IpdE1), a recently characterized subunit of a heteromeric ACAD that is specific for the bicyclic, post-ring-opening C-ring side chain, this residue is replaced with tyrosine. This residue is also seen in SCAD and MCAD, while the equivalent residue Phe is found in human very long chain ACAD .…”

“…"−" denotes instances in which this substrate was not tested. +++ indicates only specific activity has been determined for this enzyme (41 μM min −1 mg −1 ) 26. (b) Sequence similarity network encompassing steroid-degrading ACADs.…”

Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium Tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-Cholest-4,22- Dien-24-Oyl-CoA

“…Throughout the cholesterol catabolic pathway, we have identified protein architectures and assemblies that are unique to actinomycetes and Mtb. 26,30,[39][40][41][42] Importantly, chsB1 is required for the in vivo survival of Mtb in mice, 35 and the structures of ChsB1 elucidated in this work provide insight into binding site cavities that differ from its homologues and will allow discrimination between host and pathogen enzymes by inhibitors in the future. The enzymatic evolutionary adaptations that have emerged in Mtb for the breakdown of cholesterol and the in vivo essentiality of various genes in the pathway highlight the value of this pathway's enzymes for further exploitation in drug discovery.…”