Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme
Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incomin...
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KeAi Communications Co., Ltd.
2025-03-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X24001170 |
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| author | Yongjian Qiao Junbo Wang Dashan Zhang Xiaoqing Zheng Baixin Lin Yongkang Huang Yulin Liao Zixin Deng Lingxin Kong Delin You |
| author_facet | Yongjian Qiao Junbo Wang Dashan Zhang Xiaoqing Zheng Baixin Lin Yongkang Huang Yulin Liao Zixin Deng Lingxin Kong Delin You |
| author_sort | Yongjian Qiao |
| collection | DOAJ |
| description | Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives. |
| format | Article |
| id | doaj-art-54414fec2e0e4c5faa482a1153bf00ef |
| institution | OA Journals |
| issn | 2405-805X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | KeAi Communications Co., Ltd. |
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| series | Synthetic and Systems Biotechnology |
| spelling | doaj-art-54414fec2e0e4c5faa482a1153bf00ef2025-08-20T02:24:59ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2025-03-01101768510.1016/j.synbio.2024.08.006Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzymeYongjian Qiao0Junbo Wang1Dashan Zhang2Xiaoqing Zheng3Baixin Lin4Yongkang Huang5Yulin Liao6Zixin Deng7Lingxin Kong8Delin You9State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaCorresponding author.; State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaCorresponding author.; State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, ChinaAnisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.http://www.sciencedirect.com/science/article/pii/S2405805X24001170TransketolaseAromatic keto acidPromiscuityAnisomycinBiosynthesis |
| spellingShingle | Yongjian Qiao Junbo Wang Dashan Zhang Xiaoqing Zheng Baixin Lin Yongkang Huang Yulin Liao Zixin Deng Lingxin Kong Delin You Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme Synthetic and Systems Biotechnology Transketolase Aromatic keto acid Promiscuity Anisomycin Biosynthesis |
| title | Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme |
| title_full | Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme |
| title_fullStr | Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme |
| title_full_unstemmed | Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme |
| title_short | Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme |
| title_sort | biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique thdp dependent enzyme |
| topic | Transketolase Aromatic keto acid Promiscuity Anisomycin Biosynthesis |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X24001170 |
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