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.
Lin
L L
LL
Department of Food and Nutrition, Hung-Kuang Institute of Technology, Shalu, Taichung 433-01, Taiwan.
Liao
H F
HF
Chien
H R
HR
Hsu
W H
WH
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000032:analysis* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D012694:Serine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D003545:Cysteine