Structure Basis Of Aminotransferase And Isomerases From Gentamicin Biosynthesis And A Broad-Selective Acyltransferase Domain From The Polyketide Synthase

Natural products are an important source of human medicine.however,discovering new natural products with novel structures and activities are becoming less frequent,and the abuse uses of antibiotics has led to a growing problem of bacterial resistance.Therefore,clarifying the biosynthetic pathway of natural products,especially antibiotics,is imperative to improve their efficacy or drug resistance.The research object of this paper is aminoglycosides and polyketides.We have made an intensive study of the key enzymes in the biosynthetic pathways with the structural biology.The first part of this paper is to study the structural biology of transaminase GenB 1 and isomerase GenB2 in Gentamicin biosynthesis pathway.Gentamicin is a mixture with various component.The toxicity and antibacterial activity of each component are different.Obtaining the monocomponent is essential to improve the efficacy.GenB1 and GenB2 are responsible for the amination at the C’-6 position,especially the GenB2,is the key enzymes to generate C2 and C2a.Determination of their structure and mechanism is quite significant for obtaining the monocomponent of gentamicin.Firstly,we acquired high-purity GenB1 and GenB2 proteins.high-resolution crystals were obtained by crystal screening and optimization,their structures were solved by molecular replacement,and we obtained the structure of GenB2 complex with the substrate analog G418.GenB1 and GenB2 are typical Type I PLP-dependent enzymes that act as a dimer.The PLP is located in the active pocket between a small C-terminal domain and a large PLP-binding domain,where existed three conserved catalytic residues(Asp,Thr,and a Thr from in another monomer molecule)stabilizing the PLP.Compared with other Type I enzymes,GenB1 and GenB2 have a large active pocket that contains PLP and three sugar ring substrates,and there are numerous hydrophilic residues in the active pocket that interact with substratethe residues of the active pockets of GenB1 and GenB2 were found to be very conserved.The only difference is that GenB1 has a polar residue Arg71 on the N-terminal domain,while orresponding residues are changed as Phe62 in GenB2.The discrepancy may be the key reason for their functional distinct.Comparing to other Type I isomerase revealed a conservative Lys256/Tyr143 residue in the GenB2 active pocket,meaning that the GenB2 also adopts the "two-base mechanism".The second part of the dissertation is about the structural basis of the broad-selective acyltransferase domain SpnD-AT.The acyltransferase domain AT in the polyketide synthase(PKS)module is the "gateway" to identify different extender units,and modifying AT can obtain numerous non-natural compounds with different activities.SpnD-AT is an acyltransferase domain with broad-selective for various substrates,and can specifically recruit aromatic extender unit,breaking the limitation that the traditional ATs can only select limited malonyl thioestersWe first expressed the SpnD-AT and obtained the high-purity protein.The regular shape crystals were obtained by crystal screening and optimization,and collected a set of reflection data at 1.6 A.The structure of SpnD-AT was solve.d by molecular replacement.Then we soaked the crystals with two substrates(benzylmalonyl-SNAC and pentynylmalonyl-SNAC)and obtained structures complex with two substrates.The structure of SpnD-AT is Similar to other canonic AT domains,consisting of a large α/βhydrolase fold and a small ferredoxin.The substrate located at a hydrophobic interface of the α/β-hydrolase and ferredoxin subdomains where existed three conserved catalytic residues(Ser173,Arg198,and His271).The extender unit formed a covalent link with OH of the Catalytic residues Ser,and the a-carbon present an S configuration.The side chain of Phe145 formed strongπ-π interaction with the phenyl and the alkynyl group of the substrate and regarded as an important residue for the recognition of aromatic extender units.We compared the structure of SpnD-AT with erythromycin Ery-AT5,and found that SpnD-AT has a large hydrophobic active pocket which is very important for building AT specificity and activity.in Ery-AT5,there are three conserved residues Tyr278,Ser280,and Gln150 that affect the size and hydrophobicity of active pocket,which demonstrates the three residues were identified critical for restricting their substrate specificity.Engineering the residues Tyr278 and Ser280 of Ery-AT6 to flexible residues can either reverse or broaden the substrate specificity.This study pave a solid way to introduce diverse structural varieties into the polyketide carbon scaffold.