Analysis of essential amino acids in lasso peptide lariatins for anti-mycobacterial activity by single amino acid substitution
Lariatin A and B discovered as an anti-mycobacterial peptides are ribosomally synthesized by Gram positive Rhodococcus jostii. The compounds are unique cyclic peptides, consisting of 18 and 20 L-amino acid (aa) residues with an internal linkage between the α-amino group of G1 and the γ-carboxyl group of E8. The C-terminal tail passes through the ring to form the rigid 'lasso' structure. Production of lariatins in R. jostii is dependent upon a five-gene cluster, larA to larE. We established a simple production system of amino acid substitutions of lariatin by R. jostii K01-B0171ΔlarA, Using this system, we have performed mutational scanning of lariatin, constructing and analyzing about 30 singly substituted derivatives of lariatin and defined aa residues important for production of lariatin and anti-mycobacterial activity. The results showed that three aa residues (G1, R7 and E8) in the circle and three aa residues (W9, V10 and G11) in the threaded segment of the tail are important for lariatin production, and that one aa residue (Y6) in the circle and three aa residues (V10, N14 and K17) in the threaded segment of the tail are important for anti-mycobacterial activity. These findings will open the way for design and construction of more potent lariatin-based anti-mycobacterial agents.