Biology subjects

Germer, P.

Publications and source records attributed to Germer, P..

3 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↗

bioRxiv2024

Structural Insights into Broad-Range Polyphosphate Kinase 2 II Enzymes Applicable for Pyrimidine Nucleoside Diphosphate Synthesis

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP-consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85% of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49%. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

biochemistry↗

bioRxiv2023

Archaeal S-adenosyl-L-homocysteine hydrolases: structure, function and substrate preferences

S-Adenosyl-O_SCPLOWLC_SCPLOW-homocysteine hydrolase (SAHH) reversibly cleaves S-adenosyl-O_SCPLOWLC_SCPLOW-homocysteine (SAH), the product of S-adenosyl-O_SCPLOWLC_SCPLOW-methionine (SAM)-dependent methylation reactions. The conversion of SAH into adenosine and O_SCPLOWLC_SCPLOW-homocysteine (Hcy) plays an important role in the regulation of the methyl cycle. An alternative metabolic route for SAM regeneration in the extremophiles Methanocaldococcus jannaschii and Thermotoga maritima was identified with the deamination of SAH to S-inosyl-O_SCPLOWLC_SCPLOW-homocysteine (SIH). Herein, we report the first structural characterisation of different archaeal SAHHs together with a biochemical analysis of various SAHHs from all three domains of life. We found that homologues deriving from the Euryarchaeota phylum show a higher conversion rate with SIH compared to SAH. Crystal structures of SAHH originating from Pyrococcus furiosus in complex with SIH and inosine as ligands, show architectural flexibility in the active site and offer deeper insights into the binding mode of hypoxanthine-containing substrates. Altogether, the findings presented in this study support the understanding of an alternative metabolic route for SAM and offer insights into the evolutionary progression and diversification of SAHHs involved in methyl and purine salvage pathways.

biochemistry↗