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The crystal structure of glutamate dehydrogenase from Clostridium symbiosum at 0.6 nm resolution.

ABSTRACT: The structure of glutamate dehydrogenase from Clostridium symbiosum has been solved by single-crystal X-ray-diffraction studies at 0.6 nm resolution by using a combination of isomorphous replacement and molecular averaging. The electron-density map reveals that this glutamate dehydrogenase is a hexameric oligomer, arranged in 32 symmetry, of cylindrical appearance and dimensions, of length 10.8 nm and radius 4.4 nm. From an analysis of this map each subunit appears to contain some 55% alpha-helix and is organized into two distinct globular domains separated by a deep cleft. The subunits associate using the domain closest to the 32-symmetry point, making intimate contacts around the threefold and twofold interfaces. The second domain shows structural homology to the NAD-binding domain of other dehydrogenases, and difference Fourier analysis has shown that the NAD is bound in both a structurally equivalent position and a similar conformation to that observed for those related enzymes.

SUBMITTER: Rice DW

PROVIDER: S-EPMC1147779 | biostudies-other | 1987 Mar

REPOSITORIES: biostudies-other

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Project description:Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD(+)-agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40 mM glutamate and 1 mM NAD(+) at pH 8.8, or K89L/A163G/S380A with 70 mM norleucine and 1 mM NAD(+) at pH 8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC(50)=70 mM, h=2.4), and glutarate gave approx. 30-fold activation (EC(50)=35 mM, h=2.3). For the triple mutant, corresponding values were 80 mM and 2.2 for succinate, and 75 mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH 8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The "foreign" subunit evidently impedes inter-subunit communication to some extent.

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The crystal structure of glutamate dehydrogenase from Clostridium symbiosum at 0.6 nm resolution.

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