P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate

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.

Bax protein structure and related research progress of eutectic structure / 国际生物医学工程杂志

Apoptosis involves multiple signaling pathways. The intrinsic signaling pathway is the mitochondrial apoptotic pathway, which is caused by a series of processes. First, the pro-apoptotic factors such as Bax are activated by signaling molecules and transfer to the mitochondrial outer membrane forming protein pores, thus the mitochondrial membrane permeability is affected, and then the downstream caspase-9 is activated and the apoptosis initiation is induced by releasing cytochrome C. In order to explore the apoptosis initiation activated by small molecules, the specific structural changes of Bax in apoptosis were studied. The results showed that there is a hydrophobic pocket structure near the C-terminal S184 of the Bax protein. This structure can be combined with certain small molecular substances specifically remove phosphorylation S184, and regulate Bax protein to promote apoptosis activity. At present, the nuclear magnetic structure of Bax protein has been obtained and the crystal structure has not been revealed. The eutectic structure formed by corresponding with other proteins in the Bcl-2 family has been resolved, which can be used to study the interactions between proteins and to understand the specific structural changes in the formation of heterologous dimers during apoptosis, site changes, etc. In this paper, the Bax protein structure resolved by nuclear magnetic structure was reviewed to learn the change of the sites in the induced apoptosis so as to promote the research on apoptosis initiation.