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

Pyridoxal phosphate (PLP)-dependent enzymes afford access
to a
variety of non-canonical amino acids (ncAAs), which are premier building
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 γ-substituted ncAAs. Identification
of VGK enzymes for biocatalysis faces a distinct challenge because
the subfamily contains both desirable synthases and 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 γ-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 γ-addition reaction to afford
access to a unique set of γ-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.