Chemoenzymatic synthesis of SAM analogs This study highlights a broadly applicable platform for the facile syntheses of SAM analogs that is directly compatible with downstream SAM utilizing enzymes. The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with rapid SAM analog decomposition and thereby opens the door to the further interrogation of a wide range of SAM utilizing enzymes. As a proof of concept for the feasibility of natural product ‘alkylrandomization’, the coupled strategy was used to generate a small set of indolocarbazole analogs in conjunction with the rebeccamycin O-methyltransferase RebM.

S-Adenosyl-l-methionine (AdoMet) is an essential enzyme cosubstrate in fundamental biology with an expanding range of biocatalytic and therapeutic applications. In recent years, technologies enabling the synthesis and utilization of novel functional AdoMet surrogates have rapidly advanced. Developments highlighted within this brief review include improved syntheses of AdoMet analogs, unique S-adenosyl-l-methionine isosteres with enhanced stability, and corresponding applications in epigenetics, proteomics and natural product/small molecule diversification (‘alkylrandomization‘).

S-adenosyl-l-methionine (AdoMet) is an essential enzyme cosubstrate in fundamental biology with an expanding range of biocatalytic and therapeutic applications. We report the design, synthesis, and evaluation of stable, functional AdoMet isosteres that are resistant to the primary contributors to AdoMet degradation (depurination, intramolecular cyclization, and sulfonium epimerization). Corresponding biochemical and structural studies demonstrate the AdoMet surrogates to serve as competent enzyme cosubstrates and to bind a prototypical class I model methyltransferase (DnrK) in a manner nearly identical to AdoMet. Given this conservation in function and molecular recognition, the isosteres presented are anticipated to serve as useful surrogates in other AdoMet-dependent processes and may also be resistant to, and/or potentially even inhibit, other therapeutically relevant AdoMet-dependent metabolic transformations (such as the validated drug target AdoMet decarboxylase). This work also highlights the ability of the prototypical class I model methyltransferase DnrK to accept non-native surrogate acceptors as an enabling feature of a new high-throughput methyltransferase assay.

Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoMet) bound, and unbound forms. Notably, among these structures, the AdoEth-bound form offers the first MAT structure containing a non-native product and cumulatively, these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in E. coli and human. As a thermostable MAT structure from archaea, the structures herein also provide as a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed ‘alkylrandomization’ and/or the study of methylation in the context of biological processes.

A chemoenzymatic platform for the synthesis of Sadenosyl-l-methionine (SAM) analogues compatible with downstream SAM-utilizing enzymes is reported. Forty-four non-native S/Se-alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non-native SAM analogues. As a proof of concept for the feasibility of natural product "alkylrandomization", a small set of differentiallyalkylated indolocarbazole analogues was generated by using a coupled hMAT2-RebM system (RebM is the sugar C4'-Omethyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.

The structural conservation among methyltransferases (MTs) and MT functional redundancy is a major challenge to the cellular study of individual MTs. As a first step toward the development of an alternative biorthogonal platform for MTs and other AdoMet-utilizing enzymes, we describe the evaluation of 38 human methionine adenosyltransferase II-α (hMAT2A) mutants in combination with 14 non-native methionine analogues to identify suitable bioorthogonal mutant/analogue pairings. Enabled by the development and implementation of a hMAT2A high-throughput (HT) assay, this study revealed hMAT2A K289L to afford a 160-fold inversion of the hMAT2A selectivity index for a non-native methionine analogue over the native substrate L-Met. Structure elucidation of K289L revealed the mutant to be folded normally with minor observed repacking within the modified substrate pocket. This study highlights the first example of exchanging L-Met terminal carboxylate/amine recognition elements within the hMAT2A active-site to enable non-native bioorthgonal substrate utilization. Additionally, several hMAT2A mutants and L-Met substrate analogues produced AdoMet analogue products with increased stability. As many AdoMet-producing (e.g., hMAT2A) and AdoMet-utlizing (e.g., MTs) enzymes adopt similar active-site strategies for substrate recognition, the proof of concept first generation hMAT2A engineering highlighted herein is expected to translate to a range of AdoMet-utilizing target enzymes.

The effect of foul-release-coated propellers on the cathodic protection current demand was investigated in a sea trial involving a Royal Canadian Navy training vessel. The decrease in current demand was significant with a 76% reduction while underway, and up to 40% reduction while alongside. The measured current demand was consistent with that modeled using a boundary element technique. As expected, the largest contributors to ship current demand were the area of exposed hull and propellers, and ship speed.

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