The Structural Studies Of O-carbamoyltransferase (VtdB) And Pictete-spenglerase (AslB)

Secondary metabolites are various structurally complex compounds synthesized through complex secondary metabolic pathways,including vitamins,hormones,antibiotics,alkaloids,and toxins,which are closely related to people's lives.Therefore,it is of great significance to develop and modify secondary metabolites,and to study relevant synthesis pathways and important enzymatic catalytic mechanisms involved in the synthesis process.As the function and structure of enzymes are closely related,structural studies of enzymes are fundamental to revealing their function.By analyzing the crystal structure of key enzymes in the synthesis pathways of two secondary metabolites,this paper reveals its specific catalytic mechanism at the atomic level,which provides a theoretical basis for the exploitation and modification of antibiotics and ?-carboline alkaloids.(I): Venturicidin A(VTD-A)is macrolide antibiotics that can inhibit mitochondrial H+-ATPase in Escherichia coli and exerts antibacterial activity by inhibiting ATP synthesis in various fungi and bacteria.Venturicidins A also has antitrypanosomal activity and has the potential to be developed as a new antitrypanosomal drug.In addition,venturicidin A can act as antibiotic adjuvant and enhance resistance to aminoglycoside antibiotics,which may provide a new idea for targeting aminoglycoside resistance.The synthesis pathway of venturicidin A includes the formation of the skeleton and the post-modification process.In the process of post-modification,the Ocarbamoyltransferase Vtd B plays an important role.At present,the mechanism of action of Vtd B are still unclear.To determine the catalytic process of Vtd B,we cloned and expressed Vtd B protein in vitro and resolved the crystal structure of Vtd B and carbamoyladenylate complex by X-ray diffraction.An asymmetric unit of Vtd B crystal has four molecules,each bound to a magnesium ion and a carbamoyladenylate.The monomer structure contains two domains,the Kae1-like domain and the Yrd C-like domain,and both magnesium ion and carbamoyladenylate are bound to the Kae1-like domain.By structural comparison with known homologous proteins,the active pockets and possible catalytic processes of the Vtd B were identified.The active pockets of the carbamoylphosphate and venturicidin B are located inside the interface of the two domains and the Kae1-like domain,respectively.In the Kae1-like domain,a magnesium ion and carbamoyladenylate are bound near the binding pocket of venturicidin B,and the magnesium ion forms coordination bonds with H157,H161,D180,D363,and carbamoyladenylate,respectively.The comparison of the active sites of the substrates showed that the active sites of carbamoylphosphate were conserved,while the active sites of venturicidin B were not conserved.We speculated that Vtd B might recognize venturicidin B differently.(II): The ?-carboline alkaloids are an important class of alkaloids with a wide range of biological activities and have long been used in the treatment of tumours,malaria,and other diseases,so it is of great significance for the study of the mechanism of its biosynthetic pathway.In the actinomycete strain Kitasatospora setae NBRC 14216 T,the ?-carboline skeleton of the ?-carboline alkaloid Kitasetaline is catalyzed by the PictetSpenglerases(PSases)Ksl B,which inhibits the expression of glucose regulatory protein 78(GRP78)in tumour cell lines and has the potential to act as a lead compound for anticancer drugs.In the synthesis pathway of Kitasetaline,L-tryptophan and ?-ketoglutaric acid are used as starting substrates,and under the action of Ksl B,an intermediate compound kitasetalic acid is formed to complete the final synthesis.Therefore,as the first enzyme in the synthesis process,the specific molecular mechanism catalyzed by Ksl B is essential for the study of the synthesis of the ?-carboline alkaloid kitasetaline.In this study,we preliminarily studied the catalytic activity of Ksl B and its homologous protein Asl B and resolved the three-dimensional crystal structure of Asl B.Through sequence alignment and structural analysis,we speculated that Ksl B and Asl B may have the same substrate and catalytic mechanism.The overall structure of Asl B is a dimer composed of two monomers,and each monomer is composed of two domains.The binding pockets of the two substrates are located in the central region of the dimer.In the binding pocket of L-tryptophan,the catalytic activity center Glu276 recognizes the amino group of tryptophan and acts as an "acid-base" catalyst for Asl B catalytic reactions.The benzene rings of Thr263 and Phe98 can form an indole-sandwich structure to stabilize the indole-ring of tryptophan.Through structural analysis,we found that the key residues binding to L-tryptophan are relatively conserved,while the residues of another substrate ?-ketoglutaric acid binding site are completely unconserved,suggesting that Asl B may catalyze the binding of ?-ketoglutaric acid to L-tryptophan to form ?-carboline skeleton through a novel mechanism of action.