Structural And Functional Studies Of StnA In The Biosynthesis Of Antitumor Drug

Streptonigrin(STN)is an aminoquinone antibiotic produced by Streptomyces flocculus CGMCC4.1223 and was first reported in 1959.It is active against a broad range of tumors.In addition,it also shows in vivo and in vitro antiviral properties,and broad spectrum antibacterial activities against bacteria and fungi.However,its clinic use is limited because of its severe and prolonged bone narrow depression.Therefore,STN attracted much attention from chemists,pharmacists and biologists.As the upstream boundary of STN gene cluster,stnA gene encodes an?/?hydrolase which was shown efficiently to hydrolyze methyl ester of 10?-desmethoxystreptonigrin to produce10?-desmethoxystreptonigrin.10?-desmethoxystreptonigrin has higher activity and lower toxic effect than STN,and it will be used to instead of STN in clinic treatment.In order to reveal StnA functional characteristics,the crystal structure of StnA is necessary.It can help us to reveal the molecular mechanism of hydrolysis and to improve the biocatalysts in industry.In this research,wild-type StnA,selenomethionine substituted StnA and S185A mutant with substrate STM were expressed,purified,and crystallized,respectively.The crystal structures of SeMet-StnA and the S185A/STM complex were submitted to PDB,and their access numbers were 5HDF and 5HDP,respectively.The crystal structure shows that StnA has an?-helical cap domain positioned atop a common?/?hydrolase domain.The catalytic triad is Ser185-His349-Asp308 with Ser185 acting as nucleophile.The transition state stabilized by an oxyanion hole formed by the backbone amides of residues Ala102 and Leu186.This suggests that StnA is a typical?/?hydrolase and the hydrolysis mechanism is the same as other esterase.Based on the analysis of sequence,structure and phylogenetic tree,we found the main difference between StnA and other esterase.In general,the substrate is bound at an internal cavity and/or large surface cleft between the interfaces of?/?hydrolase domain and cap domain.But StnA's binding pocket is mainly composed of the cap domain.However,StnA is significantly different from the lipolytic enzymes without open and close conformations or long tunnel.We propose that StnA reveals a unique substrate binding mode.The interactions between substrate STM and StnA were identified by both dry and wet experiments,indicating 8 main hydrophobic interactions and 2 hydrogen bonds(Asn277 and Ala282).In order to confirm the results of molecular dynamics simulation,the hydrophobic residues which have interacted with STM were mutated to hydrophilic residues and the binding assay of these mutants was performed by biolayer interferometry system.A high correlation between the binding free energy and logK_D(R=0.74)was observed.However,X-ray crystallography is not suitable for dynamic process of protein.We used molecular dynamics simulation to explain that the two disulfide bonds(Cys64-Cys85 and Cys312-Cys319)play an important role in enzyme stability and maintaining the shape of binding pocket.Methyl ester of lavendamycin(LME)is also an intermediate product in the streptonigrin biosynthesis pathway,but it almost couldn't be hydrolyzed by StnA.The key hydrogen bond(Ala282)may influence on the binding,and LME might escaped from binding pocket.The electron density for the 60 residues at the N-terminal is too poor to build the structure.We speculated that StnA is a membrane-bound enzyme according to the its function,the results of bioinformatics tools online and phylogenetic analysis.The full-length StnA model was built by I-TASSER and simulated by MD.Maybe the?0 helix which is formed by No.33-50 residues firmly anchors to the membrane and two long loops between it are very flexible.The cis-Thr77 located in a long loop between?1 and?2 moved vertically with the loop in N-terminal.The community result also showed that cis-Thr77 and part of N-60 residues have strong fluctuation correlation.Then we purified full-length StnA used membrane protein protocol.After detergent treatment,the target protein was detected in the membrane fraction.Meanwhile,StnA exhibited better activity in the 10%nonionic detergent.In conclusion,structural,computational,enzymatic activity assays were combined to elucidate the molecular mechanism of StnA.The results provide an important theoretical basis and practical significance for future research of biosynthesis of antitumor drug streptonigrin.It enhanced the feasibility of StnA used as biocatalysts in industry.