Shrinking the FadE proteome of Mycobacterium tuberculosis: insights into cholesterol metabolism through identification of an α2β2 heterotetrameric acyl coenzyme A dehydrogenase family

Abstract

The ability of the pathogen Mycobacterium tuberculosis to metabolize steroids like cholesterol and the roles that these compounds play in the virulence and pathogenesis of this organism are increasingly evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. FadE26-FadE27 catalyzes the dehydrogenation of 3β-hydroxy-chol-5-en-24-oyl-CoA, an analog of the 5-carbon side chain cholesterol degradation intermediate. Genes encoding the α2β2 heterotetrameric ACAD structures are present in multiple regions of the M. tuberculosis genome, and subsets of these genes are regulated by four different transcriptional repressors or activators: KstR1 (also known as KstR), KstR2, Mce3R, and SigE. Homologous ACAD gene pairs are found in other Actinobacteria, as well as Proteobacteria. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four subpathways of cholesterol metabolism.

Fig 1

M. tuberculosis cholesterol degradation pathway. Not all individual steps are shown.

Fig 2

Operonic organization of M. tuberculosis fadE genes studied in this work. In the M. tuberculosis genome, there are six operons containing multiple genes annotated as fadE genes, all of which are regulated by cholesterol (6) except in the operon containing fadE12 and fadE13.

Fig 3

Analysis of FAD binding sites in M. tuberculosis ACAD complexes. (A) Homology model of the heterotetrameric FadE26-FadE27 complex based on the crystal structure of human i3VD (shown with the S₂ rotational axis). The FAD in a complete and conserved binding site is shown in yellow, and the FAD in a nonconserved binding site is shown in gray. The catalytic glutamate is shown in red. (B) Active site and FAD binding sites in M. tuberculosis homotetrameric and heterotetrameric ACADs compared to human i3VD (24) and MCAD (40). Conserved residues for riboflavin binding and adenosine binding are shown in green and blue, respectively, and nonconserved residues are shown in gray (also see Fig. S1 in the supplemental material).

Fig 4

Phylogenetic network for bacterial ACAD genes homologous to cholesterol-regulated and proximal operonic M. tuberculosis fadE genes. Each node (circle) represents a gene. Distances between nodes represent relative evolutionary distance between two genes. For illustrative purposes, only edges that represent distances between two genes in the same operon are shown. The pink nodes are the 11 M. tuberculosis fadE genes that are regulated by cholesterol. The green nodes are non-M. tuberculosis Actinobacteria. The yellow nodes are Proteobacteria. Gene clusters I to IV referenced in the text are indicated as I to IV, respectively, in the figure. (A) Full phylogenetic network. (B) The 11 M. tuberculosis fadE genes present in operons are highlighted. The edges are colored according to the operon coloring in Fig. 2. (C) Magnification of the fadE31-fadE32-fadE33 gene triplet. This network was generated in Cytoscape 2.8.2 using an edge-weighted, spring embedded layout (41). A high-resolution version with labeled nodes is available as Fig. S4 in the supplemental material.