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.