Crystal Structure Analysis Of Cis-prenyltransferase Subunit NUS1 From Saccharomyces Cerevisiae

The cis-prenyltransferase?EC 2.5.1.87?is a member of the isopentenyl pyrophosphate synthase superfamily and catalyzes the continuous condensation of isoprenyl pyrophosphate?IPP?to the farnesyl pyrophosphate?FPP?backbone catalyzes the formation of long-chain polyisoprene pyrophosphate.Long-chain polyisoprene pyrophosphate is dephosphorylated by phosphatase to produce polyprenyl alcohol,then its?-isopentenyl unit is reduced to form dolichol by NADPH-dependent microsomal reductase.As a glycosyl carrier,dolichol participates in the N-glycosylation of proteins and it has important significance for life activities.The cis-prenyltransferase of Saccharomyces cerevisiae consists of the heterodimeric subunit NUS1?nuclear undecaprenyl pyrophosphate synthase 1,NUS1?and the subunit RER2?Retention in the ER mutation 2,RER2?,and no related crystal structure has been reported.In this paper,the NUS1 subunit protein from Saccharomyces cerevisiae was used as research object,and its heterologous expression,mutation modification,protein purification,crystallization and crystal structure analysis were studied.In order to delete the transmembrane region of the protein and achieve soluble expression,a N-terminal 119 amino acid residues truncation mutant?N119-NUS1 was constructed.The N-terminal 28 amino acids were further truncated to construct the truncation mutant?N147-NUS1,which was no longer sensitive to TEV protease and purified to obtain a single band of protein.To obtain a single polymer protein,construct a mutation?N147-NUS1 C184A/C293A by mutating cysteineAfter the soluble protein was soluble in Escherichia coli BL21 trxB?DE3?strain,it was purified by nickel affinity chromatography and DEAE anion exchange chromatography.The N-terminal histidine tag was excised by TEV Protease and then purified by nickel affinity chromatography to obtain purified protein.After crystallization of?N147-NUS1 C184A/C293A protein,9 crystallization conditions were obtained,optimized at condition of 0.1 mol·L^-1 Sodium Cacodylate pH 6.5;0.2 mol·L^-1Ammonium Sulfate;19%?w/v?PEG 8000,protein crystals are available for structural resolution.The crystal structure model of?147-NUS1 C184A/C293A was obtained by X-ray diffraction data collection and structural modification of?N147-NUS1 C184A/C293A protein crystal.?147-NUS1 C184A/C293A crystal space group is P 2₁2₁2₁,resolution 2?,PDB code6JCN.The crystal structure of?N147-NUS1 C184A/C293A can be used as a molecular replacement template for the crystal structure analysis of similar proteins.The crystal structure of?N147-NUS1 C184A/C293A provides a crystal structure basis for the analysis of the catalytic mechanism of the heterodimer cis-prenyltransferase.By analyzing the crystal structure of?N147-NUS1 C184A/C293A and the crystal structure difference of Staphylococcus aureus undecaprenyl diphosphate synthase?UPPS?,it can provide some meaningful information for semi-rational design and simulated screening for new antibiotics.