Affiliation:
1. School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
Abstract
Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues inClostridium symbiosumNAD+-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons withE. coliNADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0. They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (∼170-fold for P262S and F238S/P262S with relatively small changes for NADH). A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8) and slightly decreased activity with NADH. At position 242, “P6” of the “core fingerprint,” where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity.
Funder
Science Foundation Ireland
Subject
Molecular Biology,Biochemistry
Cited by
6 articles.
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