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.
Genetic variation in the membrane trafficking adapter protein complex 4 (AP-4) can result in pathogenic neurological phenotypes including microencephaly, spastic paraplegias, epilepsy, and other developmental defects. We lack molecular mechanisms responsible for impaired AP-4 function arising from genetic variation, because AP-4 remains poorly understood structurally. Here, we analyze patterns of AP-4 genetic evolution and conservation to identify regions that are likely important for function and thus more susceptible to pathogenic variation.We map known variants onto an AP-4 homology model and predict the likelihood of pathogenic variation at a given location on the structure of AP-4. We find significant clustering of likely pathogenic variants located at the interface between the β4 and N-μ4 subunits, as well as throughout the C-μ4 subunit. Our work offers an integrated perspective on how genetic and evolutionary forces affect AP-4 structure and function. As more individuals with uncharacterized AP-4 variants are identified, our work provides a foundation upon which their functional effects and disease relevance can be interpreted.
The Phospholipase D (PLD) superfamily is linked to neurological disease, cancer, and fertility, and a recent report correlated a potential loss-of-function PLD2 polymorphism with hypotension. Surprisingly, PLD2 −/− mice exhibit elevated blood pressure accompanied by associated changes in cardiac performance and molecular markers, but do not have findings consistent with the metabolic syndrome. Instead, expression of endothelial nitric oxide synthase (eNOS), which generates the potent vasodilator nitric oxide (NO), is decreased. An eNOS inhibitor phenocopied PLD2 loss and had no further effect on PLD2 −/− mice, confirming the functional relationship. Using a human endothelial cell line, PLD2 loss of function was shown to lower intracellular free cholesterol, causing upregulation of HMG Co-A reductase, the rate-limiting enzyme in cholesterol synthesis. HMG Co-A reductase negatively regulates eNOS, and the PLD2-deficiency phenotype of decreased eNOS expression and activity could be rescued by cholesterol supplementation and HMG Co-A reductase inhibition. Together, these findings identify a novel pathway through which the lipid signaling enzyme PLD2 regulates blood pressure, creating implications for on-going therapeutic development of PLD small molecule inhibitors. Finally, we show that the human PLD2 polymorphism does not trigger eNOS loss, but rather creates another effect, suggesting altered functioning for the allele.
Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in regulation DIM biosynthesis.
Although the interfacial membrane protein cholesterol oxidase is structurally and kinetically well-characterized, its orientation in and mode of interaction with cholesterol-containing membranes have not been established. Cholesterol oxidase can alter the structure of the cell membrane in pathogenic bacteria and is thus a potential antimicrobial drug target. We recently developed a mass spectrometry-based isotope-coded mass tag (ICMT) labeling method to monitor the real-time solvent-accessible surface of peripheral membrane proteins, such as cholesterol oxidase. The ICMT strategy utilizes maleimide-based isotope tags that covalently react with cysteine residues. In this study, by comparing the ICMT labeling rates of cysteine variants of cholesterol oxidase, we determined which residues of the protein were engaged with the protein–lipid interface. We found that upon addition of cholesterol-containing lipid vesicles, four cysteine residues in a cluster near the substrate entrance channel are labeled more slowly with ICMT probes than in the absence of vesicles, indicating that these four residues were in contact with the membrane surface. From these data, we generated a model of how cholesterol oxidase is oriented when bound to the membrane. In conclusion, this straightforward method, which requires only microgram quantities of protein, offers several advantages over existing methods for the investigation of interfacial membrane proteins and can be applied to a number of different systems.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.