To ascertain the functional role of cysteine residue in 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase from Corynebacterium glutamicum, site-directed mutagenesis was performed to change each of the three residues to serine. Plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged DAHP synthase. Analysis of the purified wild-type and mutant enzymes by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a molecular mass of approximately 45 kDa. Cys(145)Ser mutant retained about 16% of the enzyme activity, while DAHP synthase activity was abolished in Cys(67)Ser mutant. Kinetic analysis of Cys(145)Ser mutant with PEP as a substrate revealed a marked increase in K-m with significant change in k(cat), resulting in a 13.6-fold decrease in k(cat)/K-m(PEP). Cys(334) was found to be nonessential for catalytic activity, although it is highly conserved in DAHP synthases. From these studies, Cys(67) appears important for synthase activity, while Cys(145) plays a crucial role in the catalytic efficiency through affecting the mode of substrate binding.