Isaksen, G. V. | Search bioRxiv

bioRxiv2023

Biophysical characterization and analysis of a mesophilic chorismate mutase from B. pumilus

Chorismate mutases have extensively been used as computational benchmarking systems for enzyme catalysis, yet the roles entropy and enthalpy play in catalysis are still not fully understood. Thus, it is important to better understand these enzymes for potential research or industrial applications. Here, we report the first crystal structure and kinetic characterization of a chorismate mutase from Bacillus pumilus. This enzyme exhibits a high degree of similarity to a known mesophilic chorismate mutase from Bacillus subtilis. Using this crystal structure, we further employ EVB/MD simulations to construct Arrhenius plots, allowing us to extract thermodynamic activation parameters. Overall, this study provides new insights into the structural and functional features of the B. pumilus chorismate mutase and highlights its potential as a valuable enzyme for biocatalytic and biotechnological applications.

biochemistry↗

bioRxiv2020

Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family

The first SAM degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as an S-adenosyl-L-methionine (SAM) hydrolase forming 5-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha-beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography and NMR spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis, in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.

biochemistry↗