Structure Determination of Kahalalide Analogues Based on Metagenomic Analysis of a Bryopsis sp. Marine Green Alga

Two kahalalide analogues were isolated from a Bryopsis sp. marine green alga. Even though our initial structure determination of the peptides by NMR and MS identified them as kahalalide Z(1) (KZ(1); 3) and Z(2) (KZ(2); 4), the absolute configuration of the Thr residues by Marfey's analysis was different from those found in kahalalide F (KF), 3, and 4. To ascertain the absolute configuration of the amino acid residues genetically, we conducted a metagenomic analysis for symbiotic bacteria in the alga, leading to the biosynthetic gene cluster (BGC) responsible for producing the kahalalides named kahalalides Z(3) (KZ(3); 1) and Z(4) (KZ(4); 2). The identification of amino acid residues based on the A-domain suggested these peptides possess the amino acid sequence d-allo-Thr-l-Val-l-Val-d-Val residues at the N-terminus, instead of the d-Val-l-Thr-l-Val-d-Val residues found in KF, 3, and 4. The N-terminal amino acid sequence including absolute configuration was unambiguously determined by a comparison of LCMS data of synthetic tetrapeptides and the hydrolysates derived from 1 and 2. This structural difference is caused by swapping the substrate specificities of the first two A-domains.