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Publication Abstract from PubMed

Pyridoxal phosphate (PLP)-dependent enzymes afford access to a variety of non-canonical amino acids (ncAAs), which are premier buildings blocks for the construction of complex bioactive molecules. The vinylglycine ketimine (VGK) subfamily of PLP-dependent enzymes plays a critical role in sulfur metabolism and is home to a growing set of secondary metabolic enzymes that synthesize gamma-substituted ncAAs. Identification of VGK enzymes for biocatalysis faces a distinct challenge because the subfamily contains both desirable synthases as well as lyases that break down ncAAs. Some enzymes have both activities, which may contribute to pervasive mis-annotation. To navigate this complex functional landscape, we used a substrate multiplexed screening approach to rapidly measure the substrate promiscuity of 40 homologs in the VGK subfamily. We found that enzymes involved in transsulfuration are less likely to have promiscuous activities and often possess undesirable lyase activity. Enzymes from direct sulfuration and secondary metabolism generally had a high degree of substrate promiscuity. From this cohort, we identified an exemplary gamma-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). This enzyme is thermostable and has high expression (~400 mg protein per L culture), enabling preparative scale synthesis of thioether containing ncAAs. When assayed with l-allylglycine, CahyGS catalyzes a stereoselective gamma-addition reaction to afford access to a unique set of gamma-methyl branched ncAAs. We determined high-resolution crystal structures of this enzyme that define an open-close transition associated with ligand binding and set the stage for future engineering within this enzyme subfamily.

Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family.,Zmich A, Perkins LJ, Bingman C, Acheson JF, Buller AR ACS Catal. 2023 Sep 1;13(17):11644-11655. doi: 10.1021/acscatal.3c02498. Epub , 2023 Aug 21. PMID:37720819^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.