|
|||||
|
|
Engineering the Substrate Specificity of a P450 Dimerase Enables the Collective Biosynthesis of Heterodimeric Tryptophan-Containing Diketopiperazines |
|
| Authors: | Dr Chenghai Sun Dr Bao-Di Ma Guangjun Li Wenya Tian Lu Yang Dr Haidong Peng Dr Zhi Lin Prof Dr Zixin Deng Prof Dr Xu-Dong Kong Prof Dr Xudong Qu |
| Institution: | 1. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Rd., 200240 Shanghai, China
Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, 201203 Shanghai, China These authors contributed equally to this work,;2. Abiochem Biotechnology Co. Ltd., 200240 Shanghai, China;3. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Rd., 200240 Shanghai, China Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, 201203 Shanghai, China;4. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Rd., 200240 Shanghai, China |
| Abstract: | Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in NasF5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12 self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively. |
| Keywords: | Biocatalysis Biosynthesis Dimerization P450 Protein Engineering |