Terminal alkyne amino acids can be used for Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reactions, which makes them of interest for protein-labeling applications. The soil bacterium Streptomyces cattleya synthesizes the two related terminal alkyne amino acids β-ethynylserine (βes) and l-propargylglycine (Pra), which differ in the hydroxylation of the β-carbon.

Michelle Chang at the University of California, Berkeley, and her colleagues used a comparative genomic approach to identify the genes of the βes biosynthetic pathway. They found one cluster encoding BesABCDE in S. cattleya, which is not present in Streptomyces species that do not produce βes. The authors generated S. cattleya strains in which these genes were deleted and then used comparative metabolomics experiments to identify the precursors and intermediates of the βes pathway. In the first step, the halogenase BesD chlorinates the substrate lysine to 4-Cl-lysine. BesC catalyzes the oxidative C–C cleavage of 4-Cl-lysine, yielding the alkene intermediate 4-Cl-allylglycine, which is converted to Pra by BesB. βes is not formed directly by hydroxylation of Pra. Instead, BesA catalyzes the reaction of glutamate with Pra, and the γ-Glu-Pra intermediate is formed. γ-Glu-Pra is hydroxylated by BesE, forming γ-Glu-βes, and yet unknown cellular hydrolases then release βes.

The researchers reconstituted γ-Glu-βes biosynthesis in vitro. Pra and the pathway intermediates can be produced in Escherichia coli by coexpression of the βes cluster genes. The authors demonstrated that Pra was incorporated into the E. coli proteome when they used an engineered Pra-specific aminoacyl–tRNA synthase and labeled Pra-containing protein extracts with a fluorescent dye via the CuAAC reaction.

The βes pathway enzymes enable the synthesis of halo, terminal alkene, and alkyne amino acids in cells, and these nonstandard amino acids could be incorporated into proteins by genetic code expansion for a variety of applications.