TokK and ThnK are cobalamin-dependent radical SAM enzymes that methylate a carbapenem precursor three or two times, respectively, in a sequential, non-processive fashion.

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.

TokK and ThnK stereoselectively abstract the pro-S hydrogen, and methylation proceeds with inversion of absolute configuration at C6.

The N-sulfonated monobactams harbor considerable potential to combat emerging bacterial infections that are problematic to treat due to their metallo-β-lactamase mediated resistance against conventional β-lactam antibiotics. Herein, we report a...