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Publication Abstract from PubMed

Hitachimycin is a macrolactam antibiotic with an (S)-beta-phenylalanine (beta-Phe) at the starter position of its polyketide skeleton. (S)-beta-Phe is formed from l-alpha-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 (S)-beta-Phe analogs to a DeltahitA strain. We obtained six hitachimycin analogs with F at the ortho, meta, or para position and Cl, Br, or a CH3 group at the meta 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 beta-amino acid-selective adenylation enzyme that introduces (S)-beta-Phe into the hitachimycin biosynthetic pathway. The KM values of the incorporated (S)-beta-Phe analogs and natural (S)-beta-Phe were similar. However, the KM values of unincorporated (S)-beta-Phe analogs with Br and a CH3 group at the ortho or para 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 (S)-beta-3-Br-phenylalanine sulfamoyladenosine (beta-m-Br-Phe-SA) revealed that the bulky meta-Br group is accommodated by the conformational flexibility around Phe328, whose side chain is close to the meta position. The aromatic group of beta-m-Br-Phe-SA is surrounded by hydrophobic and aromatic residues, which appears to confer the conformational flexibility that enables HitB to accommodate the meta-substituted (S)-beta-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.

Mutational Biosynthesis of Hitachimycin Analogs Controlled by the beta-Amino Acid-Selective Adenylation Enzyme HitB.,Kudo F, Takahashi S, Miyanaga A, Nakazawa Y, Nishino K, Hayakawa Y, Kawamura K, Ishikawa F, Tanabe G, Iwai N, Nagumo Y, Usui T, Eguchi T ACS Chem Biol. 2021 Feb 24. doi: 10.1021/acschembio.1c00003. PMID:33625847^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.