An Atypical Arginine Dihydrolase Involved in the Biosynthesis of Cyclic Hexapeptide Longicatenamides

• Published in: Chemistry, an Asian journal an Asian journal, 2021-06, Vol.16 (11), p.1382-1387
• Main Authors: Liu, Chao, Hashimoto, Junko, Kudo, Kei, Shin‐ya, Kazuo, Kakeya, Hideaki
• Format: Article
• Language: English
• Subjects: Amino acids; arginine dihydrolase; Biosynthesis; Chemistry; cyclic peptide; Design modifications; enzyme kinetics; Lysine; molecular docking; Peptides; Substrates
• ISSN: 1861-4728; 1861-471X
• DOI: 10.1002/asia.202100181

The incorporation of non‐proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA‐containing cyclic hexapeptides, longicatenamides A−D, were isolated using a combined‐culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22‐like proteins form a novel clade that is separated from other guanidine‐modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31‐ and 4.70‐fold that of the wild‐type (WT), respectively. In addition, characterization of the Lon20‐A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products. An atypical arginine dihydrolase (ADH), Lon22, is involved in the biosynthesis of nonribosomal cyclic peptide longicatenamides. Unlike classic ADHs, Lon22 can hydrolyze citrulline to produce ornithine. Lon22‐like proteins form a separate phylogenetic clade that is distinct from the three classes of guanidine‐modifying enzymes. Using computational simulations, we designed an Y80F mutant with higher catalytic efficiency than the wild‐type.