Study On Biosynthetic Mechanism Of Nucleoside Medicine Polyoxin And Pentostatin

Polyoxins(POL),a typically dipeptidyl nucleoside antibiotic,was widely applied to treat phytopathogenic fungi infecting vegetables,fruits and crops due to its potent anti-fungi and environmental-friendly features.Structurally,polyoxin consists of a nucleoside skeleton and two post modified amino acids,which is a competitive inhibitor of chitin synthase,as it mimics UDP-N-acetylglucosamine,a building block for chitin biosynthesis of fungi's cell wall.In the early studies,our lab has cloned the polyoxin biosynthetic gene cluster,and proposed its biosynthetic pathway with combined experimental and bioinformatics analysis.However,the detailed pathway leading to the unique nucleoside skeleton biosynthesis has long remained elusive,which aroused the research interests of scientists.One of my studies aims to clarify the biosynthetic pathway of polyoxin nucleoside skeleton at the molecular level based on previous studies,to provide a theoretical basis for discovering and creating high-activity polyoxin analogues.Based on the cosmid pJTU4774 which containing polyoxin biosynthetic gene cluster,and the host strain CXR14 in which the whole polyoxin gene cluster has been deleted,PCR-targeting strategy was performed to construct polyoxin nucleoside skeleton related genes polD polH,polI,polJ and polK mutant strain.Analysis of the fermentation products of these mutants confirmed that polD,polH,and polK deletion mutants accumulate related intermediates.We then isolated and purified these intermediates by semi-prepared HPLC,and elucidated their chemical structures through LC-MS and NMR analysis lays a solid foundation for further studies on related proteins functions.PolH belongs to Radical SAM superfamily,which always catalyze novel enzymatic reactions.Therefore,PolH plays an important role in the biosynthesis of polyoxin nucleoside skeleton.As a consequence,study PolH function is very essentially significant.However,we failed to reconstitute PolH enzymatic activity with in vitro assays using the purified intermediate as substrate.After some attempts and analysis,we confirmed that PolH can catalyze 3'-EUMP to octosyl acid.Base on this result,it not only revised the polyoxin biosynthesis but also enriched functions of Radical SAM enzyme superfamily.Pentostatin(PTN),a purine nucleoside antibiotic produced by S.antibioticus NRRL 3238 containing a unique 1,3-diazepine ring,is a powerful adenosine deaminase(ADA)inhibitor and has been clinically used to treat leukemia.Arabinofuranosyladenine(Ara-A,vidarabine),another purine nucleoside antibiotic,which also produced by S.antibioticus NRRL 3238.Structurally differs from adenosine in epimerization at the C-2' hydroxyl group,has also been clinically used as an adenosine analogue against DNA viruses infected diseases.In decades,the biosynthetic gene cluster of PTN have not been reported yet.In our previous work,we have identified the biosynthetic gene cluster of PTN,but found this gene cluster is simultaneously responsible for the biosynthesis of Ara-A,while both of their pathways are totally independent.One of my other studies based on genetics and enzymology has evidenced the biosynthetic mechanism during PTN and Ara-A biosynthesis including“single gene cluster for two independent pathways",and protect-protege strategy that PTN,as ADA inhibitor is capable of protecting Ara-A from deaminated by ADA.Mutational analysis of PTN and Ara-A related genes in cosmid pWHU1106 containing PTN and Ara-A biosynthetic gene clusters by PCR-targeting.We then introduced the pWHU1106 variants into the CXR14 leading to the corresponding recombinants.Further analysis of the metabolites of these recombinants revealed that three genes penABC are required for PTN biosynthesis,and penDEFGHIJ are involved in Ara-A biosynthesis.According to bioinformatics analysis,identified four ADA homologs from S.antibioticus.These four ADAs were individually over-expressed and purified in E.coli,and tested for their activity in vitro with adenosine and Ara-A as substrates.Result indicated that only SanADA3 is capable of deaminating both adenosine and Ara-A obviously,and PTN preferably inhibits Ara-A deamination.In addition,we measured the kinetic parameters(Km,kcat,Ki)of SanADA3 for adenosine and Ara-A,and the results are fully consistent with in vitro studies.PenB pertains to short-chain dehydrogenase/reductase(SDR)superfamily,which can catalyze 6-keto PTN conversing to PTN as the last step of PTN biosynthesis.In vitro experiments evidenced that PenB are capable of catalyzing both 6-keto PTN and PTN,and showed the last step of PTN biosynthesis is a reversible reaction.On the basis of previous work,we have studied the biosynthetic mechanism of nucleoside antibiotics polyoxin and pentostatin in vivo and in vitro.When it refers to polyoxin nucleoside skeleton,we have obtained two intermediates from the gene-knockout mutants,and demonstrated the function of Radical SAM protein PolH for C-C bond formation.As to pentostatin,we have demystified "single gene cluster for two independent pathways" and protect-protege strategy in vivo and in vitro.We also figured out the PenB could catalyze the reversible reaction at the last step of pentostatin biosynthesis.