Biochemical and structural characterization of a class A ß-lactamase from Nocardia cyriacigeorgica.

Nocardia are Gram-positive bacteria from the Actinobacteria phylum. Some Nocardia species can infect humans and are usually considered to be opportunist pathogens, as they often infect immunocompromised patients. Although their clinical incidence is low, many Nocardia species are now considered to be emerging pathogens. Primary sites of infection by Nocardia are the skin or the lungs, but dissemination to other body parts is very frequent. These disseminated infections are very difficult to treat and thus are tackled with multiple classes of antibiotics, in addition to the traditional treatment targeting the folate pathway. ß-Lactams are often included in the regimen, but many Nocardia species present moderate or strong resistance to some members of this drug class. Genomic, microbiological and biochemical studies have reported the presence of class A ß-lactamases (ABLs) in a handful of Nocardia species, but no structural investigation of Nocardia ß-lactamases has yet been performed. In this study, the expression, purification and preliminary biochemical characterization of an ABL from an N. cyriacigeorgica (NCY-1) clinical strain are reported. The crystallization and the very high resolution crystal structure of NCY-1 are also described. The sequence and structural analysis of the protein demonstrate that NCY-1 belongs to the class A1 ß-lactamases and show its very high conservation with ABLs from other human-pathogenic Nocardia. In addition, the presence of one molecule of citrate tightly bound in the catalytic site of the enzyme is described. This structure may provide a solid basis for future drug development to specifically target Nocardia spp. ß-lactamases.

Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs.

Mycobacteria have an atypical thick and waxy cell wall. One of the major building blocks of such mycomembrane is trehalose monomycolate (TMM). TMM is a mycolic acid ester of trehalose that possesses long acyl chains with up to 90 carbon atoms. TMM represents an essential component of mycobacteria and is synthesized in the cytoplasm, and then flipped over the plasma membrane by a specific transporter known as MmpL3. Over the last decade, MmpL3 has emerged as an attractive drug target to combat mycobacterial infections. Recent three-dimensional structures of MmpL3 determined by X-ray crystallography and cryo-EM have increased our understanding of the TMM transport, and the mode of action of inhibiting compounds. These structures were obtained in the presence of detergent and/or in a lipidic environment. In this study, we demonstrate the possibility of obtaining a high-quality cryo-EM structure of MmpL3 without any presence of detergent through the reconstitution of the protein into peptidiscs. The structure was determined at an overall resolution of 3.2 Å and demonstrates that the overall structure of MmpL3 is preserved as compared to previous structures. Further, the study identified a new structural arrangement of the linker that fuses the two subdomains of the transmembrane domain, suggesting the feature may serve a role in the transport process.

Crystal structure of MAB_4123, a putative flavin-dependent monooxygenase from Mycobacterium abscessus.

Numerous bacteria from different phylae can perform desulfurization reactions of organosulfur compounds. In these degradation or detoxification pathways, two-component flavin-dependent monooxygenases that use flavins (FMN or FAD) as a cofactor play important roles as they catalyse the first steps of these metabolic routes. The TdsC or DszC and MsuC proteins belong to this class of enzymes as they process dibenzothiophene (DBT) and methanesulfinate. Elucidation of their X-ray structures in apo, ligand-bound and cofactor-bound forms has provided important molecular insights into their catalytic reaction. Mycobacterial species have also been shown to possess a DBT degradation pathway, but no structural information is available on these two-component flavin-dependent monooxygenases. In this study, the crystal structure of the uncharacterized MAB_4123 protein from the human pathogen Mycobacterium abscessus is presented. The structure solved at high resolution displays high similarity to homologs from Rhodococcus, Paenibacillus and Pseudomonas species. In silico docking approaches suggest that MAB_4123 binds FMN and may use it as a cofactor. Structural analysis strongly suggests that MAB_4123 is a two-component flavin-dependent monooxygenase that could act as a detoxifying enzyme of organosulfur compounds in mycobacteria.