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Protein Science, Vol 7, Issue 4 966-974, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

A monomeric mutant of Clostridium symbiosum glutamate dehydrogenase: Comparison with a structured monomeric intermediate obtained during refolding

S. MILLEVOI, A. PASQUO, R. CHIARALUCE, V. CONSALVI, L. GIANGIACOMO, K. L. BRITTON, T. J. STILLMAN, D. W. RICE and P. C. ENGEL
Dipartimento di Scienze Biochimiche, `A. Rossi Fanelli', Universita `La Sapienza', Roma, Italy These authors have equally contributed

The refolding of Clostridium symbiosum glutamate dehydrogenase (GDH) involves the formation of an inactive structured monomeric intermediate prior to its concentration-dependent association. The structured monomer obtained after removal of guanidinium chloride was stable and competent for reconstitution into active hexamers. Site-directed mutagenesis of C. symbiosum gdh gene was performed to replace the residues Arg-61 and Phe-187 which are involved in subunit-subunit interactions, as determined by three-dimensional structure analysis. Heterologous over-expression in Escherichia coli of the double mutant (R61E/F187D) led to the production of a soluble protein with a molecular mass consistent with the monomeric form of clostridial GDH. This protein is catalytically inactive but cross-reacts with an anti-wild-type GDH antibody preparation. The double mutant R61E/F187D does not assemble into hexamers. The physical properties and the stability toward guanidinium chloride and urea of R61E/F187D were studied and compared to those of the structured monomeric intermediate.
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