Glutamate decarboxylase (GAD) catalyses the decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Improvement of the enzymatic properties of GAD is important for the low-cost synthesis of GABA. In this study, utilizing sequences of enzymes homologous with GAD from lactic acid bacteria, highly mutated GADs were designed using sequence-based protein design methods. Two mutated GADs, FcGAD and AncGAD, generated by full-consensus design and ancestral sequence reconstruction, had more desirable properties than native GADs. With respect to thermal stability, the half-life of the designed GADs was about 10 °C higher than that of native GAD. The productivity of FcGAD was considerably higher than those of known GADs; more than 250 mg/L of purified enzyme could be produced in the E. coli expression system. In a production test using 26.4 g of l-glutamate and 3.0 g of resting cells, 17.2 g of GABA could be prepared within one hour, without purification, in a one-pot synthesis.

Radical S-adenosylmethionine (RaS) enzymes catalyze some of the most fascinating transformations in Nature. With only ~100 of the >300,000 members studied to date, it is safe to assume that a plethora of new reactions and reaction mechanisms remain to be elucidated. It is by now relatively easy to spot RaS enzymes in microbial genomes. However, to determine the reactions that they carry out, detailed structural characterization of the product(s) is necessary, a process that still represents a significant roadblock in the study of RaS enzymes. We have recently combined natural products structural elucidation along with RaS enzymology to provide a proof of concept for how the confluence of these approaches can lead to the discovery of new natural products and RaS enzyme-mediated transformations. Herein, we provide guidelines for expressing, purifying, and reconstituting a subclass of RaS enzymes that contain a so-called SPASM domain, as well as characterizing the reactions that they catalyze using a combination of HR/MSn and NMR investigations. Application of these approaches will aid in expanding the chemical and biosynthetic repertoire of RaS enzymes in the future.