The ability of science and medicine to control the pathogen Mycobacterium tuberculosis (Mtb) requires an understanding of the complex host environment within which it resides. Pathological and biological evidence overwhelmingly demonstrate how the mammalian steroid cholesterol is present throughout the course of infection. Better understanding Mtb requires a more complete understanding of how it utilizes molecules like cholesterol in this environment to sustain the infection of the host. Cholesterol uptake, catabolism, and broader utilization are important for maintenance of the pathogen in the host and it has been experimentally validated to contribute to virulence and pathogenesis. Cholesterol is catabolized by at least three distinct sub-pathways, two for the ring system and one for the side chain, yielding dozens of steroid intermediates with varying biochemical properties. Our ability to control this worldwide infectious agent requires a greater knowledge of how Mtb uses cholesterol to its advantage throughout the course of infection. Herein, the current state of knowledge of cholesterol metabolism by Mtb is reviewed from a biochemical perspective with a focus on the metabolic genes and pathways responsible for cholesterol steroid catabolism.

Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases. Increasing metabolically available IP by overexpression of a bacterial phosphomevalonate decarboxylase (MPD) in Nicotiana tabacum resulted in significant enhancement in both monoterpene and sesquiterpene production. These results indicate that perturbing IP metabolism results in measurable changes in terpene products derived from both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Moreover, the unpredicted peroxisomal localization of bacterial MPD led us to discover that the step catalysed by phosphomevalonate kinase (PMK) imposes a hidden constraint on flux through the classical MVA pathway. These complementary findings fundamentally alter conventional views of metabolic regulation of terpenoid metabolism in plants and provide new metabolic engineering targets for the production of high-value terpenes in plants.

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 ␣ 2 ␤ 2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol.

Eukarya, Archaea, and some Bacteria encode all or part of the essential mevalonate (MVA) metabolic pathway clinically modulated using statins. Curiously, two components of the MVA pathway are often absent from archaeal genomes. The search for these missing elements led to the discovery of isopentenyl phosphate kinase (IPK), one of two activities necessary to furnish the universal five-carbon isoprenoid building block, isopentenyl diphosphate (IPP). Unexpectedly, we now report functional IPKs also exist in Bacteria and Eukarya. Furthermore, amongst a subset of species within the bacterial phylum Chloroflexi, we identified a new enzyme catalyzing the missing decarboxylative step of the putative alternative MVA pathway. These results demonstrate, for the first time, a functioning alternative MVA pathway. Key to this pathway is the catalytic actions of a newly uncovered enzyme, mevalonate phosphate decarboxylase (MPD) and IPK. Together, these two discoveries suggest that unforeseen variation in isoprenoid metabolism may be widespread in nature.DOI: http://dx.doi.org/10.7554/eLife.00672.001

Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis (M. tuberculosis) plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. Based on early metabolite identification studies in multiple actinomycetes, it has been proposed that cholesterol side chain metabolism requires one or more acyl-CoA dehydrogenases (ACADs). There are 35 genes annotated as encoding ACADs in the M. tuberculosis genome. Here we characterize a heteromeric ACAD encoded by Rv3544c and Rv3543c, formerly named fadE28 and fadE29, respectively. We now refer to genes Rv3544c and Rv3543c as chsE1 and chsE2 in recognition of their validated activity in cholesterol side chain dehydrogenation. Analytical ultracentrifugation and LC/UV experiments establish that ChsE1-ChsE2 forms an α2β2 heterotetramer, a new architecture for an ACAD. Our bioinformatic analysis and mutagenesis studies reveal that heterotetrameric ChsE1-ChsE2 has only two active sites. E241 in ChsE2 is required for catalysis of dehydrogenation by ChsE1-ChsE2. Steady state kinetic analysis establishes the enzyme is specific for an intact steroid ring system compared to hexahydroindanone substrates with specificity constants (kcat/KM) of 2.5 × 105 ± 0.5 s-1 M-1 vs 9.8 × 102 ± s-1 M-1 respectively, at pH 8.5. The characterization of a unique ACAD quaternary structure involved in sterol metabolism that is encoded by two distinct cistronic ACAD genes opens the way to identification of additional sterol metabolizing ACADs in M. tuberculosis and other actinomycetes through bioinformatic analysis.