Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), one of the deadliest infectious diseases. The alarming health context coupled with the emergence of resistant M. tuberculosis strains highlights the urgent need to expand the range of anti-TB antibiotics. A subset of anti-TB drugs in use are prodrugs that require bioactivation by a class of M. tuberculosis enzymes called Baeyer-Villiger monooxygenases (BVMOs), which remain understudied. To examine the prevalence and the molecular function of BVMOs in mycobacteria, we applied a comprehensive bioinformatic analysis that identified six BVMOs in M. tuberculosis, including Rv3083 (MymA), Rv3854c (EthA), Rv0565c, and Rv0892, which were selected for further characterization. Homology modeling and substrate docking analysis, performed on this subset, suggested that Rv0892 is closer to the cyclohexanone BVMO, while Rv0565c and EthA are structurally and functionally similar to MymA, which is by far the most prominent type I BVMO enzyme. Thanks to an unprecedented purification and assay optimization, biochemical studies confirmed that all four BVMOs display BV-oxygenation activity. We also showed that MymA displays a distinctive substrate preference that we further investigated by kinetic parameter determination and that correlates with in silico modeling. We provide insights into distribution of BVMOs and the structural basis of their substrate profiling, and we discuss their possible redundancy in M. tuberculosis, raising questions about their versatility in prodrug activation and their role in physiology and infection. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of death worldwide. The rise in drug resistance highlights the urgent need for innovation in anti-TB drug development. Many anti-TB drugs require bioactivation by Baeyer-Villiger monooxygenases (BVMOs). Despite their emerging importance, BVMO structural and functional features remain enigmatic. We applied a comprehensive bioinformatic analysis and confirmed the presence of six BVMOs in M. tuberculosis, including MymA, EthA, and Rv0565c-activators of the second-line prodrug ethionamide-and the novel BVMO Rv0892. Combining in silico characterization with in vitro validation, we outlined their structural framework and substrate preference. Markedly, MymA displayed an enhanced capacity and a distinct selectivity profile toward ligands, in agreement with its catalytic site topology. These features ground the molecular basis for structure-function comprehension of the specificity in these enzymes and expand the repertoire of BVMOs with selective and/or overlapping activity for application in the context of improving anti-TB therapy.
Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies.
Abstract The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacyl catalysis intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme’s active site. Knowledge about the molecular details of the substrate binding site allowed us to boost the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.
La tuberculose causee par Mycobacterium tuberculosis (Mtu), est un probleme de sante majeur aggrave par la resurgence inquietante de souches de Mtu multiresistantes aux antibiotiques. Il est crucial de developper de nouvelles molecules antituberculeuses et de decouvrir des cibles therapeutiques. L'enveloppe de Mtu, atypique et tres peu permeable, contient des acides mycoliques, acides gras a tres longues chaines, qui sont essentiels et dont le metabolisme represente un reservoir de cibles validees. La degradation de ces molecules, qui semble importante lors des mecanismes de l'infection de l'hote, reste encore peu exploree.Le projet de these a porte sur l'etude de la premiere etape-clef de cette voie, catalysee par une enzyme de la famille des Baeyer-Villiger-Mono-Oxygenase (BVMO). Cette etape d'oxygenation permet d'adresser l'acide mycolique vers la voie de degradation. Nous avons selectionne cinq candidats potentiels pour cette etape sur le genome de Mtu, dont trois codant des proteines BVMO-like, presentant une sequence signature BVMO. L'expression et l'optimisation de la production des differentes proteines candidates dans deux systemes d'expression, Escherichia coli et Mycobacterium smegmatis, ont ete realisees. L'activite BVMO et les specificites de substrat des proteines candidates ont ete testees. Les candidats BVMO-like ont une plus forte activite en presence d'un substrat aliphatique presentant un pseudo-motif mycolique par rapport a d'autres substrats cyclique ou aliphatiques testes. Ces resultats sont en accord avec l'hypothese que ces candidats BVMO-like pourraient etre impliques dans la premiere etape de degradation des acides mycoliques. Deux des trois BVMO sont deja connues pour activer la prodrogue ethionamide, un antituberculeux de seconde ligne. Ces travaux montrent egalement que la troisieme BVMO peut elle aussi metaboliser ce compose.Dans le but d'approfondir les connaissances sur le role de ces proteines candidates chez Mtu, deux strategies complementaires ont ete adoptees, la production et l'analyse de (i)souches de surexpression et (ii) mutants de deletion. L'analyse lipidomique des souches de surexpression montre des variations quantitatives significatives des glycolipides contenant des acides mycoliques, le monomycolate de trehalose et le dimycolate de trehalose. D'autre part, les differents mutants de deletion presentent des variations quantitatives d'un compose apolaire potentiellement apparente a un produit de la voie de degradation des acides mycoliques. L'ensemble des travaux de lipidomique semble confirmer le lien entre les BVMO et le metabolisme des acides mycoliques, et notamment leur degradation.
ABSTRACT The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme’s active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.
