Crystallization and preliminary X-ray analysis of the NADP-specific glutamate dehydrogenase fromNeurospora crassa

Abstract

The NADP-linked glutamate dehydrogenase from Neurospora crassa has been crystallized by the hanging-drop method of vapour diffusion in the presence of 0.1 M glutamate. The crystals are trigonal and are in space group P3121 with unit-cell dimensions of a = b = 196.6, c = 102.0A and with a trimer in the asymmetric unit. A full structure determination of this enzyme will lead to an understanding of the molecular basis of inter-allelic complementation observed with hybrid hexamers of naturally occurring mutants.

The structural basis of proteolytic activation of bovine glutamate dehydrogenase

“…Chemical modification experiments (Colman and Frieden 1966;Price and Radda 1969;Coffee et al 1971) uniformly showed that the response to purine nucleotides was associated with the C-terminal 50 amino acids of the protein, a finding that correlated well with the finding that homologous GDHs of prokaryotes and fungi lacked both the 50-amino acid section and the regulation by GTP/ADP (Wootton et al 1974;Mattaj et al 1982). Latterly, the recent solution of the threedimensional structure of bovine GDH (Peterson and Smith 1999) has revealed that these 50 amino acids form an ''antenna'' protruding from the main body of the hexameric structure and totally missing in various other solved GDH structures (Rice et al 1987;Stillman et al 1995). This antenna is suggested to have been ''exapted'' from its Reprint requests to: Paul C. Engel original function linking fatty acid oxidation to amino acid catabolism in Ciliates (Allen et al 2004).…”

NADP‐dependent glutamate dehydrogenase: a dispensable function in ectomycorrhizal fungi

“…The putative gain of NADP‐GDH function is represented by a double bar according to Léjohn (1971). The fungal species mentioned are those for which a NADP‐GDH function was demonstrated, either in the present work, or in previous studies: Debaryomyces hansaneii (Alba‐Lois et al ., 2004), Schizosaccharomyces pombe (Perysinakis et al ., 1994), Penicillium chrysogenum (Diez et al ., 1999), Kluyveromyces lactis (Romero et al ., 2000), Schawanniomyces occidentalis (De Zoysa et al ., 1991), Neurospora crassa (Stillman et al ., 1995), Saccharomyces cerevisiae (Deluna et al ., 2001), Aspergillus nidulans (Hawkins et al ., 1989), Pleurotus ostreatus (Chakraborty et al ., 2003), Schizophyllum commune (Dennen & Niederpruem, 1967), Coprinus cinereus (Al‐Gharawi & Moore, 1977), Agaricus bisporus (Schaap et al ., 1996). The branch lengths are arbitrary.…”

“…We confirmed the presence of an active NADP‐GDH in the ECM ascomycetes C. geophilum and T. borchii , as already demonstrated for the ascomycetes Debaryomyces hansaneii (Alba‐Lois et al ., 2004), Schizosaccharomyces pombe (Perysinakis et al ., 1994), Penicillium chrysogenum (Diez et al ., 1999), Kluyveromyces lactis (Romero et al . 2000), Schawanniomyces occidentalis (De Zoysa et al ., 1991), N. crassa (Stillman et al ., 1995), S. cerevisiae (Deluna et al ., 2001), and Aspergillus nidulans (Hawkins et al ., 1989). For basidiomycetes, we found that some of them exhibited NADP‐GDH activity ( H. cylindrosporum and L. bicolor ) as stated by Léjohn (1971), and as already found for Pleurotus ostreatus (Chakraborty et al .…”

Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus gluatamate dehydrogenase

“…Given these dramatic differences in thermal stability, it was hoped that the structure analysis of these proteins might advance our understanding of the molecular basis of thermal stability. Previous work has led to the successful crystallisation of the glutamate dehydrogenases from the mesophiles Clostridium symbiosum (Cs) [17], Escherichia coli (Ec) [18] and Neurospora crassa (Nc) [19] and the hyperthermophile Pyrococcus furiosus (Pf) [20]. The structure determination of the Cs GIuDH has been reported at 1.9 A [6,21] and more recently that of the Pf enzyme has been completed allowing a comparison of the molecular structures of the hyperthermophilic and mesophilic enzymes [22].…”