Biomimetic S-Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes.

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

A bicyclic S-adenosylmethionine regeneration system applicable with different nucleosides or nucleotides as cofactor building blocks.

The ubiquitous cofactor S-adenosyl-l-methionine (SAM) is part of numerous biochemical reactions in metabolism, epigenetics, and cancer development. As methylation usually improves physiochemical properties of compounds relevant for pharmaceutical use, the sustainable use of SAM as a methyl donor in biotechnological applications is an important goal. SAM-dependent methyltransferases are consequently an emerging biocatalytic tool for environmentally friendly and selective alkylations. However, SAM shows undesirable characteristics such as degradation under mild conditions and its stoichiometric use is economically not reasonable. Here, we report an optimised biomimetic system for the regeneration of SAM and SAM analogues consisting of effective nucleoside triphosphate formation and an additional l-methionine regeneration cycle without by-product accumulation. The bicyclic system uses seven enzymes, S-methylmethionine as methyl donor and a surplus of inorganic polyphosphate, along with catalytic amounts of l-methionine and cofactor building block reaching conversions of up to 99% (up to 200 turnovers). We also show that the cycle can be run with cofactor building blocks containing different purine and pyrimidine nucleobases, which can be fed in at the nucleoside or nucleotide stage. These alternative cofactors are in turn converted to the corresponding SAM analogues, which are considered to be a key for the development of bioorthogonal systems. In addition to purified enzymes, the bicyclic system can also be used with crude lysates highlighting its broad biocatalytic applicability.

Loss of a conserved salt bridge in bacterial glycosyl hydrolase BgIM-G1 improves substrate binding in temperate environments.

Salt bridges are the strongest electrostatic interactions in proteins. They substantially contribute to a protein's structural stability. Thus, mutations of salt bridges are typically selected against. Here, we report on the evolutionary loss of a highly conserved salt bridge in the GH1 family glycosyl hydrolase BglM-G1. BglM-G1's gene was found in the bacterial metagenome of a temperate, seasonally cold marine habitat. In BglM-G1, arginine 75 is replaced by a histidine. While fully retaining ß-glucosidase activity, BglM-G1 is less heat stable than an H75R variant, in which the salt bridge was artificially re-introduced. However, the K m toward its substrates was lower in wild type, leading to an overall higher catalytic efficiency. Our results indicate that this loss of the salt bridge leads to higher flexibility in BglM-G1's active site, trading structural stability at high temperatures, a trait not needed in a temperate, seasonally cold habitat, for a more effective catalytic activity.