Bayer-Villiger-Monooxygenases (BVMOs) are a unique class of oxygenases playing a role in microbial degradation of diverse substrates including fatty acid derivatives. With their versatility, BVMOs offer a valuable biotechnological tool, however their physiological role remains poorly understood. Bacterial BVMOs are widespread across actinomycetes including mycobacteria, a genus that includes the important pathogen, Mycobacterium tuberculosis (Mtb). The recent collaborative study of the consortium found six BVMOs in Mtb and characterized four of them using a combination of bioinformatic, homology modelling and biochemical characterization. The study confirmed that all four enzymes exhibit BVMO activity, and highlighted their distinct substrate selectivity. Yet, physiological substrates of BVMO are still unknown. To gain molecular insights, we have performed AI-based structural analyses that revealed two BVMO subclasses with distinct substrate pockets. In addition, we optimized the purification of one BVMO that yielded a preliminary cryo-EM structure, consistent with predictions.
Mycobacteria are characterized by a wealth of long-chain fatty acids in their envelope and whose metabolism is likely related to BVMO activity. Our first studies showed the capacity of Mtb to grow on long-chain fatty acids as the sole carbon source, specifically implicating one of the six BVMO. Here, we propose to address these questions and test the hypothesis that BVMOs function as regulators of lipid degradation using integrated structural and functional insights. The project combines multiple approaches to: (i) characterize structure-function of BVMO in both subclasses, and (ii) elucidate their role in long chain fatty acid degradation. Integrated biochemical, biophysical and lipidomics analyses should allow to assess substrate specificity, metabolic impact, providing key insights into mycobacterial physiology and lipid homeostasis opening to new strategies against tuberculosis.