Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family

Pyridoxal phosphate (PLP)-dependent enzymes afford accessto avariety of non-canonical amino acids (ncAAs), which are premier buildingblocks for the construction of complex bioactive molecules. The vinylglycineketimine (VGK) subfamily of PLP-dependent enzymes plays a criticalrole in sulfur metabolism and is home to a growing set of secondarymetabolic enzymes that synthesize & gamma;-substituted ncAAs. Identificationof VGK enzymes for biocatalysis faces a distinct challenge becausethe subfamily contains both desirable synthases and lyases that breakdown ncAAs. Some enzymes have both activities, which may contributeto pervasive mis-annotation. To navigate this complex functional landscape,we used a substrate multiplexed screening approach to rapidly measurethe substrate promiscuity of 40 homologs in the VGK subfamily. Wefound that enzymes involved in transsulfuration are less likely tohave promiscuous activities and often possess undesirable lyase activity.Enzymes from direct sulfuration and secondary metabolism generallyhad a high degree of substrate promiscuity. From this cohort, we identifiedan exemplary & gamma;-synthase from Caldicellulosiruptorhydrothermalis (CahyGS). This enzymeis thermostable and has high expression (& SIM;400 mg protein perL culture), enabling preparative-scale synthesis of thioether containingncAAs. When assayed with l-allylglycine, CahyGS catalyzes a stereoselective & gamma;-addition reaction to affordaccess to a unique set of & gamma;-methyl-branched ncAAs. We determinedhigh-resolution crystal structures of this enzyme that define an open-closetransition associated with ligand binding and set the stage for futureengineering within this enzyme subfamily.

Automated summary

Pyridoxal phosphate (PLP)-dependent enzymes provide access to a variety of non-canonical amino acids (ncAAs), which are crucial for building complex bioactive molecules. The vinylglycineketimine (VGK) subfamily of PLP-dependent enzymes, known for its role in sulfur metabolism, includes both desirable synthases and lyases that break down ncAAs. Through substrate multiplexed screening, we identified a thermostable and highly expressive & gamma;-synthase from Caldicellulosiruptorhydrothermalis (CahyGS) that enables preparative-scale synthesis of thioether-containing ncAAs.