Nair, S. K. | bioRxiv Science

bioRxiv2020

Biochemical Basis for the Regulation of Biosynthesis of Antiparasitics by Bacterial Hormones

Diffusible small molecule microbial hormones drastically alter the expression profiles of antibiotics and other drugs in actinobacteria. For example, avenolide (a butenolide) regulates production of avermectin, derivatives of which are used in the treatment of river blindness and other parasitic diseases. Butenolides and {gamma}-butyrolactones control production of pharmaceutically important secondary metabolites by binding to TetR family transcriptional repressors. Here, we describe a concise, 22-step synthetic strategy for the production of avenolide. We present crystal structures of the butenolide receptor AvaR1 in isolation, and in complex with avenolide, as well as AvaR1 bound to an oligonucleotide derived from its operator. Biochemical studies guided by the co-crystal structures enable identification of 90 new actinobacteria that may be regulated by butenolides, two of which are experimentally verified. These studies provide a foundation for understanding regulation of microbial secondary metabolite production, which may be exploited for the discovery and production of novel medicines.

biochemistry↗

bioRxiv2019

Functional interactions between post-translationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans

Methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six post-translationally modified amino acids near the active site. Recent studies have identified genes that install two of these modifications (thioglycine and 5-(S)-methylarginine), yet little is known about the installation and function of the remaining post-translationally modified residues. Here, we provide in vivo evidence that a dedicated SAM-dependent methyltransferase encoded by a gene we designated mcmA is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue plays an important role in adaptation to a mesophilic lifestyle. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. Surprisingly, high-resolution crystal structures of the various unmodified MCRs were indistinguishable from the fully modified enzyme, suggesting that interactions between the post-translationally modified residues do not exert a major influence on the physical structure of the enzyme, but rather serve to fine-tune the activity and efficiency of MCR.

microbiology↗