Publication
Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia-lyases (PALs) have been previously engineered to access a range of substituted phenylalanines and heteroarylalanines, but their substrate scope remains limited, typically including only arylacrylic acids. Moreover, the enantioselectivity in the hydroamination of electron-deficient substrates is often poor. Here, we report the structure-based engineering of PAL from Planctomyces brasiliensis (PbPAL), enabling preparative-scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide- and ester-containing fumaric acid derivatives. Through the elucidation of cryo-electron microscopy (cryo-EM) PbPAL structure and screening of the structure-based mutagenesis library, we identified the key active site residue L205 as pivotal for dramatically enhancing the enantioselectivity of hydroamination reactions involving electron-deficient substrates. Our engineered PALs demonstrated exclusive alpha-regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts was further demonstrated by a preparative-scale hydroamination yielding tert-butyl protected l-aspartic acid, widely used as intermediate in peptide solid-phase synthesis.|A structure-based engineering of phenylalanine ammonia-lyase from Planctomyces brasiliensis (PbPAL) is conducted for preparative-scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide- and ester-containing fumaric acid derivatives. Our engineered PbPALs exhibit exclusive alpha-regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts is demonstrated by a preparative-scale hydroamination yielding tert-butyl protected l-aspartic acid, widely used as intermediate in peptide solid-phase synthesis. image