Chorismate synthase from Staphylococcus aureus

[摘要] The aroC gene encoding chorismate synthase and the ndk gene encoding nucleoside diphosphate kinase were cloned from Staphylococcus aureus by complementation of the Aro- phenotype of the aroC E. coli strain, GLW40. Two partial open reading frames (ORFs) encoding 3-dehydroquinate synthase and a protein which had similarity with the putative gerCC gene product from B. subtilis were also cloned. In S. aureus the aroC gene is likely to form the first gene in an operon which includes the aroB and aroA genes. It has been demonstrated in this study that the ndk genes and the gerCAgerCBgerCC genes in S. aureus are situated upstream from the aroCaroB genes; this gene organisation has also been observed in B. subtilis. The S. aureus aroC gene was expressed from the T7 promoter on plasmid pTB361. This expression system resulted in the accumulation of very high levels of soluble S. aureus chorismate synthase and facilitated the purification of the enzyme to near homogeneity, producing 100mg of enzyme from 13g of cells. No detectable immunological crossreactivity was observed between S. aureus chorismate synthase and antibodies raised against E. coli chorismate synthase. This contrasts with other chorismate synthases and indicates that there are structural differences between the chorismate synthases from S. aureus and E. coli. S. aureus chorismate synthase was determined to be a homotetramer using gel filtration and chemical crosslinking. The pH optimum determined for S. aureus chorismate synthase was found to be non-symmetrical in MOPS buffer with an optimum of activity around pH 7.0. The apparent Km for EPSP of the S. aureus enzyme was calculated to be 12.7muM and the apparent Km for FMN was calculated to be 4.8muM. The apparent Km value for FMN for the S. aureus enzyme is two orders of magnitude greater compared to other chorismate synthases, excluding the B. subtilis enzyme. S. aureus chorismate synthase was investigated using pre-steady state kinetics and a flavin intermediate was observed during turnover with a difference spectrum resembling that obtained with the E. coli enzyme. The spectral characteristics of the S. aureus flavin intermediate were different, however, with respect to its maxima, minima and its overall shape. The rate of decay (6.5s-1) of the intermediate was eight times slower than that observed for the E. coli enzyme (52s-1) and this compares well with a 7 fold lower Vmax. Tyrosine 121 of S. aureus chorismate synthase was changed to phenylalanine or alanine using site-directed mutagenesis. The conversion to alanine resulted in a loss of activity while the phenylalanine mutant retained 10% of wild-type activity.

[发布机构] University:University of Glasgow

[关键词] Molecular biology