Crystal Structural Analyses Of Herqueinone Synthetase PhnH From Penicillium Herquei

Five membered cyclic ethers are important structural elements,which present in many natural products and synthetic compounds.There are several routes could generate cyclic ethers,including oxidative cyclisation,oxa-Michael addition,epoxide opening and hydroalkoxylation.Among them,hydroalkoxylation is a powerful and efficient way of forming cyclic ethers in synthetic chemistry.Hydroalkoxylation had not been detected enzymatically until a recent investigation of the biosynthesis of the fungal metabolite herqueinone from Penicillium hequei NRRL 1040.The enzyme PhnH catalyzes enantioselective hydroalkoxylation reaction to generate the fused dihydrofuran heterocycle present in herqueinone.Here,the enzyme PhnH was expressed and purified in engineering strains and the PhnH structure were determinated.The results of PhnH bioinformatics analysis showed that there was a signal peptide.The gene encoding phnH without the N-terminal signal peptide was cloned into the pET-32a?+?vector and expressed in Escherichia coli BL21?DE3?.Protein expression in E.coli BL21?DE3?was induced by 0.5 mmol·L^-1 isopropyl?-D-thiogalactopyranoside?IPTG?at 25?for 18 h.The PhnH expressed in E.coli BL21?DE3?was purified with Ni-NTA column,TEV protease digestion and secondary Ni-NTA column.The crystallization conditions of PhnH were screened and optimized.The X-ray diffraction datasets of PhnH was collected successfully?1.33?and 1.44??at National Synchrotron Radiation Research Center?NSRRC?.The crystal structure of PhnH from PEG 4000 condition was diffracted to a high resolution of 1.33?and belonged to space group P2₁?unit-cell parameters a=69.57?,b=55.11?,c=70.97??.The crystal structures of PhnH from the two crystal forms were solved by molecular replacement.The structure of PhnH shows a tetramer.The tetramer status of PhnH was investigated by using size exclusion chromatography.The overall structure of PhnH shows a typical?-barrel fold,contains nine antiparallel?-strands and a 3₁₀0 helix??-strand?which form a cavity that may accommodate substrate.The electron density of an acetate molecule was clearly seen in the cavity of PhnH,which derived from ammonium acetate crystallization condition.There is an?-helix existed between strand?6 and?7 in bacterial proteins while a short loop in the equivalent region of PhnH.