Mutational Biosynthesis of Hitachimycin Analogs Controlled by the β-Amino Acid-Selective Adenylation Enzyme HitB.

Hitachimycin is a macrolactam antibiotic with an ()-β-phenylalanine (β-Phe) at the starter position of its polyketide skeleton. ()-β-Phe is formed from l-α-phenylalanine by the phenylananine-2,3-aminomutase HitA in the hitachimycin biosynthetic pathway. In this study, we produced new hitachimycin analogs via mutasynthesis by feeding various ()-β-Phe analogs to a Δ strain. We obtained six hitachimycin analogs with F at the , , or position and Cl, Br, or a CH group at the position of the phenyl moiety, as well as two hitachimycin analogs with thienyl substitutions. Furthermore, we carried out a biochemical and structural analysis of HitB, a β-amino acid-selective adenylation enzyme that introduces ()-β-Phe into the hitachimycin biosynthetic pathway. The values of the incorporated ()-β-Phe analogs and natural ()-β-Phe were similar. However, the values of unincorporated ()-β-Phe analogs with Br and a CH group at the or position of the phenyl moiety were high, indicating that HitB functions as a gatekeeper to select macrolactam starter units during mutasynthesis. The crystal structure of HitB in complex with ()-β-3-Br-phenylalanine sulfamoyladenosine (β--Br-Phe-SA) revealed that the bulky Br group is accommodated by the conformational flexibility around Phe328, whose side chain is close to the position. The aromatic group of β--Br-Phe-SA is surrounded by hydrophobic and aromatic residues, which appears to confer the conformational flexibility that enables HitB to accommodate the -substituted ()-β-Phe. The new hitachimycin analogs exhibited different levels of biological activity in HeLa cells and multidrug-sensitive budding yeast, suggesting that they may target different molecules.