Biology subjects

Lackner, G.

Publications and source records attributed to Lackner, G..

4 recordsLinked to original sources

bioRxiv2024

MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria.. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the {Delta}mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

microbiology↗

bioRxiv2021

Diversification by CofC and control by CofD govern biosynthesis and evolution of coenzyme F420 and its derivative 3PG-F420

Coenzyme F420 is a microbial redox cofactor that is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-D-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive. Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule, revealing its binding mode and suggesting a substrate channeling mechanism to CofD. The latter enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. Taken together, this work closes important knowledge gaps and opens up perspectives for the discovery, enhanced biotechnological production, and engineering of coenzyme F420 derivatives in the future. ImportanceThe microbial cofactor F420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F420 (3PG-F420) was discovered, enabling the production and use of F420 in heterologous hosts. By analyzing the crystal structure of a CofC homolog whose substrate choice leads to formation of 3PG-F420, we defined amino acid residues governing the special substrate selectivity. A diagnostic residue enabled reprogramming of the substrate specificity, thus mimicking the evolution of the novel cofactor derivative and successfully guiding the identification of further 3-PG-activating enzymes. Furthermore, a labile reaction product of CofC was revealed that has not been directly detected so far and CofD was shown to provide as another layer of specificity of the combined CofC/D reaction, thus controlling the initial substrate choice of CofC. The latter finding resolves a current debate in the literature about the starting point of F420 biosynthesis in various organisms.

microbiology↗

bioRxiv2019

Biosynthesis of the redox cofactor mycofactocin comprises oligoglycosylation by MftF in Mycolicibacterium smegmatis

Mycofactocin (MFT) is a redox cofactor involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. In recent years, a preliminary biosynthetic model of MFT has been established by in-vitro studies, while the final structure of MFT remained elusive. Here, we report the discovery of MFT by metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Structure elucidation revealed that MFT is decorated with up to nine {beta}-1,4-linked glucose residues. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the cofactor function of MFT by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results close an important gap of knowledge, will guide future studies into the physiological roles of MFT in bacteria and may inspire its utilization as a biomarker or potential drug target to combat mycobacterial diseases.

microbiology↗

bioRxiv2019

Metabolic pathway rerouting in Paraburkholderia rhizoxinica evolved long-overlooked derivatives of coenzyme F420

Coenzyme F420 is a specialized redox cofactor with a highly negative redox potential. It supports biochemical processes like methanogenesis, degradation of xenobiotics or the biosynthesis of antibiotics. Although well-studied in methanogenic archaea and actinobacteria, not much is known about F420 in Gram-negative bacteria. Genome sequencing revealed F420 biosynthetic genes in the Gram-negative, endofungal bacterium Paraburkholderia rhizoxinica, a symbiont of phytopathogenic fungi. Fluorescence microscopy, high-resolution LC-MS, and structure elucidation by NMR demonstrated that the encoded pathway is active and yields unexpected derivatives of F420 (3PG-F420). Further analyses of a biogas-producing microbial community showed that these derivatives are more widespread in nature. Genetic and biochemical studies of their biosynthesis established that a specificity switch in the guanylyltransferase CofC re-programmed the pathway to start from 3-phospho-D-glycerate, suggesting a rerouting event during the evolution of F420 biosynthesis. Furthermore, the cofactor activity of 3PG-F420 was validated, thus opening up perspectives for its use in biocatalysis. The 3PG-F420 biosynthetic gene cluster is fully functional in Escherichia coli, enabling convenient production of the cofactor by fermentation.

biochemistry↗