P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate.
10.1016/j.apsb.2023.03.021
- Author:
Songya ZHANG
1
;
Lin ZHANG
2
;
Anja GREULE
3
;
Julien TAILHADES
3
;
Edward MARSCHALL
3
;
Panward PRASONGPHOLCHAI
4
;
Daniel J LENG
4
;
Jingfan ZHANG
5
;
Jing ZHU
1
;
Joe A KACZMARSKI
6
;
Ralf B SCHITTENHELM
3
;
Oliver EINSLE
2
;
Colin J JACKSON
7
;
Fabrizio ALBERTI
4
;
Andreas BECHTHOLD
8
;
Youming ZHANG
1
;
Manuela TOSIN
4
;
Tong SI
1
;
Max J CRYLE
3
Author Information
1. CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
2. Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg 79104, Germany.
3. Department of Biochemistry and Molecular Biology, the Monash Biomedicine Discovery Institute, Monash University, Clayton 3800, VIC, Australia.
4. Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.
5. School of Life Sciences, University of Warwick, Gibbet Hill Campus, Coventry CV4 7AL, UK.
6. Research School of Chemistry, the Australian National University, Acton 2601, ACT, Australia.
7. ARC Centre of Excellence for Innovations in Peptide and Protein Science, Clayton 3800, VIC, Australia.
8. Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg 79104, Germany.
- Publication Type:Journal Article
- Keywords:
Cytochrome P450;
Enzyme mechanism;
Natural products;
Non-ribosomal peptide synthetase;
Peptide antibiotic;
Protein crystal structure
- From:
Acta Pharmaceutica Sinica B
2023;13(8):3561-3574
- CountryChina
- Language:English
-
Abstract:
WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,β-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.
