L-Asparagine methyl ester

Screening was carried out for microorganisms able to produce N-(l-alpha-l-aspartyl)-l-phenylalanine methyl ester [APM] from l-isoasparagine and l-phenylalanine methyl ester hydrochloride. Of the 422 strains examined, 44 strains belonging to the family Enterobacteriaceae were found to produce APM. The enzyme catalyzing APM production was purified and identified as dipeptidase E.

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min(-1) mg(-1)·protein and 136 ± 6 nmol min(-1) mg(-1)·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa.