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- Title
Identification of Essential Cysteine Residues in 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from Corynebacterium glutamicum.
- Authors
Lin, Long-Liu; Liao, Hui-Fen; Chien, Hungchien Roger; Hsu, Wen-Hwei
- Abstract
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.
- Subjects
CYSTEINE proteinases; BROMELIN; CORYNEBACTERIUM; BREVIBACTERIUM; POLYACRYLAMIDE gel electrophoresis; ZONE electrophoresis
- Publication
Current Microbiology, 2001, Vol 42, Issue 6, p426
- ISSN
0343-8651
- Publication type
Article
- DOI
10.1007/s002840010242