RESUMO
The lipoyl cofactor plays an integral role in several essential biological processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addition to the [4Fe-4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe-4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe-S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the molecular mechanism is incompletely understood. Herein, using protein-protein interaction and kinetic assays as well as site-directed mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addition of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Catálise , Proteínas de Escherichia coli/química , Ferro/química , Proteínas Ferro-Enxofre/química , Domínios Proteicos , Enxofre/químicaRESUMO
Tuberculosis (TB) is caused by infection with the bacterium Mycobacterium tuberculosis (Mtb), which primarily infects the lungs but can also cause extrapulmonary disease. Both the disease outcome and the pathology of TB are driven by the immune response mounted by the host. Infection with Mtb elicits inflammatory host responses that are necessary to control infection, but can also cause extensive tissue damage when in excess, and thus must be precisely balanced. In particular, excessive recruitment of neutrophils to the site of infection has been associated with poor control of Mtb infection, prompting investigations into the roles of neutrophils in TB disease outcomes. Recent studies have revealed that neutrophils can be divided into subpopulations that are differentially abundant in TB disease states, highlighting the potential complexities in determining the roles of neutrophils in Mtb infection. Specifically, neutrophils can be separated into normal (NDN) and low-density neutrophils (LDNs) based on their separation during density gradient centrifugation and surface marker expression. LDNs are present in higher numbers during active TB disease and increase in frequency with disease progression, although their direct contribution to TB is still unknown. In addition, the abundance of LDNs has also been associated with the severity of other lung infections, including COVID-19. In this review, we discuss recent findings regarding the roles of LDNs during lung inflammation, emphasizing their association with TB disease outcomes. This review highlights the importance of future investigations into the relationship between neutrophil diversity and TB disease severity.