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Chorismate synthase from Staphylococcus aureus

Malcolm J. Horsburgh^1,

¹Division of Biochemistry & Molecular Biology, Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
²Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland
³Department of Infection Research, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

⁴Author for correspondence: John R. Coggins. Tel: +44 141 330 5267. Fax: +44 141 330 4620. e-mail: J.Coggins@bio.gla.ac.uk

ABSTRACT

The aroC gene encoding chorismate synthase and the ndk gene encoding nucleoside diphosphate kinase (Ndk) were cloned from Staphylococcus aureus. DNA sequencing suggests that aroC is located in an operon with aroB and aroA and encodes a protein of 388 amino acids with 61% identity to the aroF gene product of Bacillus subtilis. The ndk gene of S. aureus encodes a protein of 149 amino acids which exhibits a high degree of identity to other bacterial Ndk proteins. The 3' end of the S. aureus gerCC gene was also identified by sequencing and was located immediately upstream of ndk. The gerCA and gerCB genes were found to be located upstream of gerCC by Southern hybridization analysis. This observed linkage of the gerC genes with the ndk, aroC and aroB genes has been similarly observed in B. subtilis. The S. aureus chorismate synthase was overexpressed to a high level in Escherichia coli using a T7 promoter plasmid construct, the enzyme was purified to near homogeneity in two steps and found to be a homotetramer with a subunit molecular mass, estimated by electrospray mass spectrometry, of 43024 Da. The properties of S. aureus chorismate synthase are compared with those of the B. subtilis and E. coli enzymes.

Present address: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.