Engineering a PLP-dependent Mannichase

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are versatile biocatalysts known for their ability to form diverse C-C, C-N, and C-S bonds. Despite this catalytic diversity, a PLP-dependent enzyme capable of promoting an intermolecular Mannich reaction to access α,β-diamino acids has not been described. Here, we report the engineering of LolT, a PLP-dependent Mannich cyclase from the loline alkaloid biosynthetic pathway, into a synthetically valuable Mannichase. Using iterative site-saturation mutagenesis and a double high-throughput screening platform, we identified mutations that significantly enhance LolT's non-native Mannichase activity. The best-performing variant, LolT^V4, exhibited a>60-fold improvement in catalytic turnover and enabled one-step, enantioselective synthesis of the unusual amino acid L-tambroline on a gram scale. This chapter provides a detailed experimental workflow for constructing mutant libraries, performing high-throughput functional screening, and validating hits through biochemical and analytical methods. Our work establishes a blueprint for repurposing PLP enzymes toward non-natural transformations, broadening the scope of biocatalysis for medicinal and synthetic chemistry applications.