Biology subjects

Mohr, M. K. F.

Publications and source records attributed to Mohr, M. K. F..

6 recordsLinked to original sources

bioRxiv2026

S-Adenosyl-D-methionine as a Non-Physiological Substrate for a Wide Range of SAM-Dependent Enzymes

The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine [D-SAM, (SS,RC)-SAM] instead of the native cofactor S-adenosyl-L-methionine [L-SAM, (SS,SC)-SAM] remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the nucleophilic carbon of the substrate, along with enzyme and substrate flexibility. In addition to conventional MTs, the radical SAM glutamine C-MT QCMT showed low but detectable methylation activity with D-SAM. Furthermore, the azetidine-2-carboxylic acid synthase AzeJ not only uses D-SAM but also incorporates the stereocentre of D-methionine into the cyclic amino acid product. The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropyl-1-carboxylic acid synthase (ACCS) also showed detectable turnover with D-SAM. These findings broaden the understanding of enzyme stereoselectivity, provide an overview of D-SAM-utilising enzymes, and identify first enzyme systems that may serve as starting points for engineering efforts aimed at shifting cofactor preference towards D-SAM.

biochemistry↗

bioRxiv2025

(R)-S-Adenosyl-L-methionine hydrolases counter sulfonium epimerisation in thermophilic archaea

S-Adenosyl-L-methionine (SAM) is the second most used enzyme cofactor and vital for numerous cellular reactions such as methylation or polyamine synthesis. While most stereocentres of the biologically active (SS,SC)-SAM are fixed, epimerisation at the methyl sulfonium centre is driven by heat, yielding biologically inactive (RS,SC)-SAM. This SAM diastereomer disturbs SAM-dependent pathways, posing a metabolic threat especially to thermophilic organisms. In vitro analysis shows that SAM hydrolases cleave the biologically inactive (RS,SC)-SAM, thereby constituting to a metabolic salvage pathway. For further analysis of the biological relevance, we characterised two archaeal SAM hydrolases from the thermophilic Sulfolobus acidocaldarius and the halophilic Haloferax volcanii, confirming their selectivity towards (RS,SC)-SAM in vitro. Genetic manipulation in the native hosts supports a significant role of the SAM-hydrolases in decreasing the share of intracellular (RS,SC)-SAM to sustain cellular functions in thermophilic organisms.

biochemistry↗

bioRxiv2024

Rewiring Escherichia coli to Transform Formate into Methyl Groups

BackgroundBiotechnological applications are steadily growing and have become an important tool to reinvent the synthesis of chemicals and pharmaceuticals for lower dependence on fossil resources. In order to sustain this progression, new feedstocks for biotechnological hosts have to be explored. One-carbon (C1-)compounds, including formate, derived from CO2 or organic waste are accessible in large quantities with renewable energy, making them promising candidates. Previous studies showed that introduction of the formate assimilation machinery from Methylorubrum extorquens into Escherichia coli allows assimilation of formate into the established biotechnological host. Applying this established route for formate assimilation, we here investigated utilisation of formate for the production of value-added building blocks in E. coli using S-adenosylmethionine (SAM)-dependent methyltransferases. ResultsWe first analysed methylation activity in E. coli BL21 with a two-vector system to produce three different methyltransferases together with the formate assimilation machinery. Feeding isotopically labelled formate, products with 51 - 81% 13C-labelling could be obtained by maintaining in vivo methylation activity. Focussing on improvement of in vivo methylation, we analysed two further E. coli strains with an engineered C1-metabolism and, following condition optimisation, achieved a doubled methylation activity with a share of more than 70% formate-derived methyl groups. ConclusionsThis study demonstrates the efficient transformation of formate into methyl groups in E. coli. Our findings support that feeding formate can improve the availability of usable C1-compounds and, as a result, increase in vivo methylation activity in engineered E. coli.

biochemistry↗

bioRxiv2023

Expanding the Substrate Scope of N- and O-Methyltransferases from Plants for Chemoselective Alkylation

Methylation reactions are of significant interest when generating pharmaceutically active molecules and building blocks for other applications. Synthetic methylating reagents are often toxic and unselective due to their high reactivity. S-Adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) present a chemoselective and environmentally friendly alternative. The anthranilate N-MT from Ruta graveolens (RgANMT) is involved in acridone alkaloid biosynthesis, methylating anthranilate. Although it is known to methylate substrates only at the N-position, the closest relatives with respect to amino acid sequence similarities of over 60% are O-MTs catalysing the methylation reaction of caffeate and derivatives containing only hydroxyl groups (CaOMTs). In this study, we investigated the substrate range of RgANMT and a CaOMT from Prunus persica (PpCaOMT) using compounds with both, an amino- and hydroxyl group (aminophenols) as possible methyl group acceptors. For both enzymes, the reaction was highly chemoselective. Furthermore, generating cofactor derivatives in situ enabled the transfer of other alkyl chains onto the aminophenols, leading to an enlarged pool of products. Selected MT reactions were performed at a preparative biocatalytic scale in in vitro and in vivo experiments resulting in yields of up to 62%.

biochemistry↗

bioRxiv2022

Biomimetic S-adenosylmethionine regeneration for nucleophilic and radical alkylation reactions and aminopropyltransfer

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 an efficient one-pot system 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.

biochemistry↗

bioRxiv2021

The archaeal triphosphate tunnel metalloenzyme SaTTM defines structural determinants for the diverse activities in the CYTH protein family

CYTH is a large protein superfamily that is conserved in all three domains of life with its unique triphosphate tunnel metalloenzyme (TTM) fold. Besides phosphatase functions, e.g. as RNA triphosphatase, inorganic polyphosphatase or thiamine triphosphatase, some CYTH orthologs cyclize nucleotide triphosphates to 3,5-cyclic nucleotides. So far, archaeal CYTH proteins are annotated as adenylyl cyclases although experimental evidence is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our initial in silico studies suggested a close relationship between archaeal CYTH enzymes and class IV adenylyl cyclases compared to the other CYTH-subclasses, but biochemical data showed no cyclic nucleotide production. Instead, our structural and functional analyses show a classical TTM behavior. The Ca2+-inhibited Michaelis complex indicates a two-metal ion reaction mechanism analogous to other TTMs. Different co-crystal structures of SaTTM further reveal conformational dynamics in SaTTM, let us to assume feedback inhibition in TTMs due to tunnel closure in the product state. Combining our structural insights with sequence-similarity network based in silico analysis, we further set out a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases. Major highlights- CyaB-like class IV adenylyl cyclase homologs in archaea are triphosphatases. - The co-crystal structure of SaTTM in sulfate and triphosphate bound state revealed conformational transition of the TTM tunnel during catalysis. - Atomic insights into TTM inhibition by calcium and pyrophosphate. - In silico and structure-function analysis revealed the molecular determinant for functional diversification among CYTH proteins.

biochemistry↗