Affiliation:
1. Department of Chemistry Tokyo Institute of Technology 2-12-1 Ookayama Meguro-ku Tokyo 152-8551 Japan
2. Graduate School of Agricultural and Life Sciences The University of Tokyo 1-1-1 Yayoi Bunkyo-ku Tokyo 113-8657 Japan
3. Collaborative Research Institute for Innovative Microbiology The University of Tokyo 1-1-1 Yayoi Bunkyo-ku Tokyo 113-8657 Japan
4. Present address Graduate School of Integrated Pharmaceutical and Nutritional Sciences University of Shizuoka Shizuoka 422-8526 Japan
Abstract
AbstractAdenylation enzymes catalyze the selective incorporation of aminoacyl building blocks in the biosynthesis of nonribosomal peptides and related natural products. Although β‐amino acid units are one of the important aminoacyl building blocks in natural product biosynthesis, very little is known about the engineering of β‐amino acid adenylation enzymes. In this study, we engineered the substrate specificity of the (S)‐β‐phenylalanine adenylation enzyme, HitB, involved in the biosynthesis of macrolactam polyketide hitachimycin. Based on the previously determined structure of HitB wild‐type, we mutated Phe328 and Ser293, which are located near the meta and ortho position of the (S)‐β‐phenylalanine moiety, respectively. As a result, the HitB F328V and F328L mutants efficiently activated meta‐substituted (S)‐β‐phenylalanine analogs, and the HitB T293G and T293S mutants efficiently activated ortho‐substituted (S)‐β‐phenylalanine analogs. Structural analysis of the HitB F328L and T293G mutants with the corresponding nonhydrolyzable intermediate analogs revealed an enlarged substrate binding pocket for (S)‐β‐phenylalanine analogs, providing detailed insights into the structural basis for creating enzyme substrate promiscuity. Our findings may be useful for production of various β‐amino acid‐containing natural product analogs.
Funder
Noda Institute for Scientific Research