Crystal Structural Studies Of McbC,AslB And VtdB Related To The Synthesis Of Secondary Metabolites In Streptomyces

Secondary metabolites refer to a class of small molecular substances with various chemical structures and activities which synthesized by microorganisms or plants,such as antibiotics,toxins and Hormones.Most of the secondary metabolites play a crucial role in many aspects of our lives like antibacterial,antiviral,antitumor,agricultural development and medica Health.Recent studies show that most of the secondary metabolites are synthesized by Actinomycetes,and the synthesis process is catalyzed by multi-step enzymatic reactions.Therefore,understanding the synthesis pathway of secondary metabolites and studying the catalytic mechanism of related enzymes are essential for the function of these compounds and their roles in various bio.logical processes.In this thesis,we focus on the structure and catalytic mechanism of three enzymes which involved in the secondary metabolites synthesis.Aromatic amino acid decarboxylase McbC,Pictet-Spenglerase AslB and O-carbamoyltransferase VtdB are important for the biosynthesis of Venturicidin A andβ-carboline alkaloids in Streptomyces,which are the important source of drug biosynthesis.By studying the native and substrate binding structures of these enzymes,we aim to explore the catalytic mechanism of these three enzymes and to understand the catalytic pathways of the related secondary metabolites.Further,these results may improve the industry production of antibacterial,antiviral,anticancer drugs,etc.（Ⅰ）Aromatic amino acid decarboxylase（EC 4.1.1.28）McbC catalyzes decarboxylation reactions of L-tryptophan,L-tyrosine,L-phenylalanine or its derivatives.The decarboxylation reactions are facilitated by the cofactor pyridoxal 5-phosphate（PLP）.The products tryptamine and tyramine are involved in the synthetize pathway of secondary metabolites Marinacarbolines A-D（MCBs）.To resolve the structure McbC,the plate-shaped protein crystal of McbC was successfully obtained in 0.15 M Magnesium chloride Hexahydrate,0.1 M Tris p H8.0,3.6 M 1,6-Hexanediol.We also predict the 3D structures of McbC,and McbC complexed with the cofactor pyridoxal5-phosphate（PLP）by Alpha Fold 2 and Auto Dock Tools.The predicted structures reveal that McbC is a tetramer composed of three domains:an N-terminal domain,a C-terminal domain,and a PLP binding domain.The cofactor PLP is bound in the active pocket and interacts with several key amino acids,including Asn86,Gly148,Lys149,His187,His326,and Lys327,to facilitate the decarboxylation reaction.（Ⅱ）Pictet-Spenglerase（EC 1.5.1.24）AslB catalyzes the Pictet-Spengler reaction of L-tryptophan and aldehyde or ketone molecules.Its productβ-carboline skeleton is involved in the synthesis of secondary metabolitesβ-carboline alkaloids.In this thesis,the crystal structure of AslB at 2.6（?）resolution is resolved by molecular replacement.The structure shows that AslB is a dimer composed of N-terminal domain and C-terminal domain.The binding site of substrate L-tryptophan are conserved,but the binding sites of ketoacids or aldehydes are variable.In addition,the binding model of AslB complexed with the potential substrate ketoacidsα-ketoglutarate is predicted using the software Auto Dock Tools.The structural analysis reveals that the C-terminal domain of AslB interacts withα-ketoglutarate through three specific amino acids,including Thr263,Ser260,and Ser227,which are not conserved.The result suggests thatα-ketoglutarate specifically binds to the C-terminal domain of AslB.（Ⅲ）O-carbamoyltransferase VtdB（EC 2.1.3.3）catalyzes the transfer reaction of the carbamoyl groups to ATP forming the carbamoyladenylate which is the intermediate of macrolide antibiotic Venturicidin A（Vent A）.In this thesis,the crystal structures of VtdB and VtdB complexed with carbamoyladenylate(VtdB^CAO)are determined by molecular replacement.The VtdB structure consists of Kael-like domain and Yrd C-like domain.The magnesium ion is bound to Kael-like domain and coordinate with the intermediate carbamoyladenylate.The binding model of magnesium ion will assists in the transfer process of formyl group.The reaction intermediate carbamoyladenylate is observed in the Kael-like domain and immobilized by amino acid residues His35,Asp180,Gln182,Ala213,Glu231,Gly335,Asn339 and the magnesium ion.In addition,the predict model of VtdB^{Vent B}shows that Vent B was bound in the Kael-like domain,interacting with VtdB^CAOthrough residues Arg273,Asn228 and His35,which are not conserved.The result suggests that Vent B is the specific substrate for VtdB^CAO.By studying the structures of these enzymes related to the secondary metabolites synthesize,we identify the characteristics of their active pockets and the interaction modes with substrates.Based on the catalytic mechanisms,we have got a deeper understanding of their specific roles in the production of secondary metabolites.