TY - JOUR
T1 - P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate
AU - Zhang, Songya
AU - Zhang, Lin
AU - Greule, Anja
AU - Tailhades, Julien
AU - Marschall, Edward
AU - Prasongpholchai, Panward
AU - Leng, Daniel J.
AU - Zhang, Jingfan
AU - Zhu, Jing
AU - Kaczmarski, Joe A.
AU - Schittenhelm, Ralf B.
AU - Einsle, Oliver
AU - Jackson, Colin J.
AU - Alberti, Fabrizio
AU - Bechthold, Andreas
AU - Zhang, Youming
AU - Tosin, Manuela
AU - Si, Tong
AU - Cryle, Max J.
N1 - Funding Information:
Yongwei Zhao (Monash) for assistance with binding assays; Dr Cleidiane Zampronio (School of Life Sciences, Warwick) for assistance with LC‒HRMS n Orbitrap Fusion analyses; Dr Lijiang Song (Warwick Chemistry) for preliminary MS data acquired on a BrukerMaXis Impact instrument; Prof. James de Voss (University of Queenland) for helpful discussions. This work was supported by the BBSRC (MIBTP studentship to Daniel J. Leng); the Monash Warwick Alliance (Seed Fund Award to Manuela Tosin and Max J. Cryle); the University of Warwick (Career Support Award to Manuela Tosin); Monash University, EMBL Australia, the Australian Research Council (Discovery Project DP210101752 to Max J. Cryle) and the National Health and Medical Research Council (APP1140619 to Max J. Cryle). This research was conducted by the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science ( CE200100012 ) and funded by the Australian Government. This research was funded by the National Natural Science Foundation of China ( 82104044 to Songya Zhang), the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-PTJS-003-07); We appreciate the assistance from Prof. Fu Yan (Shandong University) and Prof. Wei Zhang (Shandong University) for the P450 protein measurement. We thank the staff at beamline X06SA of the Swiss Light Source (Villigen, CH) for excellent assistance with diffraction data collection.
Funding Information:
To investigate the role of the F250 in P450Sas we constructed an altered producer strain that lacked sas16 and complemented it in trans with a variety of sas16 mutants focussed around the F250 residue. Indeed, mutation of this unusual F250 residue (together with mutating combinations of neighbouring I-helix residues, see Supporting Information Fig. S6A) into the canonical threonine residue (taken from the sequence of OxyD) in S. asterosporus showed a complete loss of biosynthetic production of WS9326A (Fig. S6B). Low levels of WS9326L (the linear form of WS9326A containing Tyr-2 residue instead of Dht) biosynthetic intermediates and WS9326B (WS9326A containing a Tyr-2 residue instead of Dht) were detected as products in these mutant strains. This matches the results from the strain in which P450Sas (Sas16) has been deleted (Fig. 3A and Supporting Information Table S5‒S7). In addition, a possible intermediate WS9326O (the linear dipeptide intermediate containing Tyr-2 residue instead of Dht) was detected from the mutant Δsas16 (Table S5 and Supporting Information Fig. S16). These results suggest that the F250T mutant of P450Sas and related mutants are no longer catalytically competent for Dht formation. Expression and purification of the P450Sas F250T mutant under the same conditions as the WT enzyme showed that this mutant is soluble and is apparently correctly folded (as evidence by P450 formation upon reduction and CO complexation), supporting this hypothesis (Fig. S7). Overall, the structural analysis of P450Sas confirmed the general P450 fold as anticipated, with comparisons to other P450 structures showing strongest similarities to P450s involved in late-stage macrolide oxidation as opposed to PCP-interacting P450s (Supporting Information Table S8). This, combined with the unusual F250 residue, suggested a hydrophobic substrate and one that was unlikely to be an aminoacyl-PCP. To further explore this hypothesis, we next expressed and purified a module tridomain (A-Mt-PCP) construct of the Sas NRPS, first confirming that A-domain was able to activate Tyr and load this onto the PCP domain (Fig. S8)32. Reconstitution of the Mt domain using SAM then led to the methylation of the PCP-bound Tyr residue, with no appreciable double methylation despite the stalled nature of the assembly line in this case. Both Tyr- and N-Me-Tyr loaded NRPS constructs were then used to explore binding and possible oxidation of tyrosyl-PCP by Sas16. Neither substrate showed any binding response or activity with P450Sas, which supports the analysis of the structure indicating an alternate substrate to an aminoacyl-PCP.Yongwei Zhao (Monash) for assistance with binding assays; Dr Cleidiane Zampronio (School of Life Sciences, Warwick) for assistance with LC‒HRMSn Orbitrap Fusion analyses; Dr Lijiang Song (Warwick Chemistry) for preliminary MS data acquired on a BrukerMaXis Impact instrument; Prof. James de Voss (University of Queenland) for helpful discussions. This work was supported by the BBSRC (MIBTP studentship to Daniel J. Leng); the Monash Warwick Alliance (Seed Fund Award to Manuela Tosin and Max J. Cryle); the University of Warwick (Career Support Award to Manuela Tosin); Monash University, EMBL Australia, the Australian Research Council (Discovery Project DP210101752 to Max J. Cryle) and the National Health and Medical Research Council (APP1140619 to Max J. Cryle). This research was conducted by the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012) and funded by the Australian Government. This research was funded by the National Natural Science Foundation of China (82104044 to Songya Zhang), the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-PTJS-003-07); We appreciate the assistance from Prof. Fu Yan (Shandong University) and Prof. Wei Zhang (Shandong University) for the P450 protein measurement. We thank the staff at beamline X06SA of the Swiss Light Source (Villigen, CH) for excellent assistance with diffraction data collection.
Publisher Copyright:
© 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences
PY - 2023/8
Y1 - 2023/8
N2 - 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.
AB - 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.
KW - Cytochrome P450
KW - Enzyme mechanism
KW - Natural products
KW - Non-ribosomal peptide synthetase
KW - Peptide antibiotic
KW - Protein crystal structure
UR - http://www.scopus.com/inward/record.url?scp=85152740762&partnerID=8YFLogxK
U2 - 10.1016/j.apsb.2023.03.021
DO - 10.1016/j.apsb.2023.03.021
M3 - Article
AN - SCOPUS:85152740762
SN - 2211-3835
VL - 13
SP - 3561
EP - 3574
JO - Acta Pharmaceutica Sinica B
JF - Acta Pharmaceutica Sinica B
IS - 8
ER -