Modular coenzyme specificity: A domain‐swopped chimera of glutamate dehydrogenase

Abstract

Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD(+)-specific) and Escherichia coli (EcGDH) (NADP(+)-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain I) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V(max)) in both directions than either of the parent enzymes from which it is constructed.

Structure of NADP+‐dependent glutamate dehydrogenase from Escherichia coli – reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

“…Domain I, which mediates the 32 symmetry contacts and is responsible for substrate binding, comprises residues 1-203 and 425-447. Domain II (residues 204-424) is responsible for cofactor binding, as recently confirmed by the construction of an active chimaeric enzyme comprising Domain I of CsGDH and Domain II of EcGDH and exhibiting NADP + specificity [22]. EcGDH and CsGDH contain an extra α-helix (α1) at the N-terminus relative to other bacterial enzymes such as PaGDH (Fig.…”

Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase

“…The primers used for generating these mutants are listed in Table S1. Mutations were confirmed with DNA sequencing and the expression constructs of these mutants were transformed into ΔGDH E. coli BL21 (DE3) (56). Expression, purification and activity assays of the mutant enzymes were performed following the same procedures as described for recombinant wild type AnGDH.…”

Overexpression in a non-native halophilic host and biotechnological potential of NAD+-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014

“…The pET5a vector was obtained from Novagen. E. coli BL21DE3 Dgdh strain was kindly provided by Dr. Michael Sharkey (Sharkey and Engel 2009) and E. coli XL10 Gold strain from Stratagene was grown in LB medium at 37°C (Sambrook et al 1989) with the addition of 100 lg/ml ampicillin for cells containing the plasmid.…”