Identification of Essential Cysteine Residues in 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from Corynebacterium glutamicum

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. Cys145Ser mutant retained about 16% of the enzyme activity, while DAHP synthase activity was abolished in Cys67Ser mutant. Kinetic analysis of Cys145Ser mutant with PEP as a substrate revealed a marked increase in Km with significant change in kcat, resulting in a 13.6-fold decrease in kcat/KmPEP. Cys334 was found to be nonessential for catalytic activity, although it is highly conserved in DAHP synthases. From these studies, Cys67 appears important for synthase activity, while Cys145 plays a crucial role in the catalytic efficiency through affecting the mode of substrate binding.