The three-dimensional crystal structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.
The structure of NAD+-dependent glutamate dehydrogenase (GDH) of ('l~i.stridium symbroszim has been solved at high resolution for the free protein [I] and for its complexes with NAD' [I] and glutamate [2] allowing an account of the structural basis of specificity [2,3] and catalysis [2] which is being systematically tested by site directed mutagenesis [4-61. This account is applicable to other members of the GDH family [7] in view of the high degree of conservation of key structural features. There are, however, unanswered questions with regard to the hnctional properties specific to clostridial GDH, notably in terms of its behaviour at alkaline pH, where it undergoes timedependent, inactivating conformational change [8] and where the measurable activity does not show [9] the shift in specificity towards monocarboxylic acid substrates seen, for example, in bovine GDH [lo] The search for a structural basis for these hnctional differences has directed attention to Asp 114. This is adjacent in the primary sequence [ 1 I , 121 to Lys 1 13, one of the three essential lysine residues identified by chemical modification [U], now shown to be involved in recognition of the a-COO-of the substrate glutamate [ 2 1 In all other reported GDH sequences this position is occupied by a neutral residue, usually asparagine or cysteine.In the glutamate complex of clostridial GDH [2], the side-chain of Asp 114 lies buried behind the side-chain of the substrate glutamate In this position the carboxyl group of the aspartate side-chain is apparently electrostatically unsatisfied by any nearby positive charge. Furthermore it appears to make only one hydrogen bond to a hydrogen bond donor (the side-chain nitrogen of QI 10) and is otherwise close to the main chain carbonyl oxygen atoms of two other conserved residues (GI22 and K89) Given the unexplained features of the pH dependence of clostridial GDH it appears possible that D1 14 might be protonated and that its charge state may be significant in determining kinetic behaviour. To explore these issues we have constructed the mutant D114N derived from the cloned wild type clostridial GDH gene [ 121 DI 14N was made by replacing the wild-type aspartate codon (GAC) with an asparagine codon (AAC) using site-directed mutagenesis with mismatch oligonucleotides [ 141 The mutated gene was subcloned into the ptac vector and the mutation confirmed by double stranded DNA sequencing [ 151 The results of IPTG induction show that a protein with electrophoretic characteristics of wild-type GDH is heavily overexpressed.Chromatography immobilised Red Remazol GG gave a highly efficient purification, as with wild-type GDH [ 161 The success of this procedure is usually diagnostic of enzyme variants in which no major structural disruption has occurred Preliminary hnctional studies show the specific activities of the wild-type and the DI 14N mutant in a standard oxidative deamination assay to be within 15% of each other at pH 7.0 (0. IM Tris-HCI). At pH 8 0 however, the specific activity of the wild-type enzy...
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