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

Two kahalalide analogues were isolated from a sp. marine green alga. Even though our initial structure determination of the peptides by NMR and MS identified them as kahalalide Z (KZ ; ) and Z (KZ ; ), the absolute configuration of the Thr residues by Marfey's analysis was different from those found in kahalalide F (KF), , and . 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 (KZ ; ) and Z (KZ ; ). The identification of amino acid residues based on the A-domain suggested these peptides possess the amino acid sequence d- -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, , and . 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 and . This structural difference is caused by swapping the substrate specificities of the first two A-domains.