D-Ornithine coopts pyrrolysine biosynthesis to make and insert pyrroline-carboxy-lysine

Abstract

D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.

Site-specific protein modifications through pyrroline-carboxy-lysine residues

“…Pyl was discovered about ten years ago as the 22nd genetically encoded proteinogenic amino acid (13)(14)(15)(16). Unexpectedly, we discovered that addition of D-Orn to growth media in the presence of the Pyl biosynthesis genes results in the generation and incorporation of a demethylated Pyl analog, Pcl (20,21) (Fig. 1) rather than Pyl as had been suggested previously (24).…”

“…Preliminary results from low oxygen fermentations in E. coli suggest that Pyl incorporation is intrinsically more efficient than Pcl (20). However, under standard expression conditions, the activity of PylB, the enzyme responsible for the methylation of the pyrrolysine precursor, or a required cofactor appears to limit the production of Pyl proteins in mammalian and E. coli cells (20).…”

“…However, under standard expression conditions, the activity of PylB, the enzyme responsible for the methylation of the pyrrolysine precursor, or a required cofactor appears to limit the production of Pyl proteins in mammalian and E. coli cells (20).…”

“…In Methanosarcinae, the TAG codon, a canonical termination signal, is primarily repurposed to signal Pyl incorporation (18). Two of the three Pyl biosynthetic genes, pylC and pylD, along with the pyrrolysl-tRNA/tRNA synthetase pair can be transferred to Escherichia coli (19) as well as mammalian cells (20), and facilitate the incorporation of pyrroline-carboxy-lysine (Pcl) rather than Pyl when D-ornithine (D-Orn) is added to the growth media ( Fig. 1) (20,21).…”

“…Two of the three Pyl biosynthetic genes, pylC and pylD, along with the pyrrolysl-tRNA/tRNA synthetase pair can be transferred to Escherichia coli (19) as well as mammalian cells (20), and facilitate the incorporation of pyrroline-carboxy-lysine (Pcl) rather than Pyl when D-ornithine (D-Orn) is added to the growth media ( Fig. 1) (20,21). Here we show that Pcl can be incorporated into a broad range of target proteins at TAG codons in both E. coli and mammalian cells, and we report a specific conjugation chemistry that enables coupling of synthetic compounds to Pcl in recombinant proteins.…”