The partial amino acid sequence of the NAD -dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity

Lilley, Kathryn S.; Baker, Patrick J.; Britton, K.L.; Stillman, Timothy J.; Brown, Paul E.; Moir, Arthur J. G.; Engel, Paul C.; Rice, David W.; Bell, John; Bell, Evelyn T.

doi:10.1016/0167-4838(91)90001-g

Cited by 39 publications

(27 citation statements)

References 32 publications

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“…Nevertheless, it is clear that similar folding patterns are achieved by relatively different sequences. It is also apparent that some of the bacterial enzymes [6,16, 401 contain short segments that more closely resemble the animal sequences, e.g. a6 and as in Peptostreptococcus usaccharolyticus and both clostridia (Fig.…”

Section: An Nadp I'gimentioning

confidence: 99%

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The glutamate dehydrogenase gene of Clotridium symbiosum

Teller

Smith

McPherson

et al. 1992

European Journal of Biochemistry

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105

101

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The gene encoding the NAD +-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M,) 49 295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-XaaXaa-Ala motif in the NADf-binding domain, which is more typical of the NADPf-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate sitedirected mutagenesis.The NAD +-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum catalyses the first step in the hydroxyglutarate pathway of glutamate fermentation 11, 21. This anaerobe is an abundant source of GDH, which amounts to approximately 1 5 % of the total cell protein during growth with glutamate. The enzyme has been purified and extensively characterized (3 -51 and peptides representing about 75% of the primary structure have been sequenced 161. In addition, C. symbiosum GDH has been crystallized in a form suitable for high-resolution X-ray analysis and the structure has been solved to 0.196 nm [7-lo]. The cloning of the gdh gene and the determination of its nucleotide sequence are important for deducing the complete amino acid sequence of the enzyme, for permitting a full interpretation of the molecular structure and for designing site-directed mutagenesis experiments. Conventional approaches for cloning the gdh gene of C . symbiosum

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Section: An Nadp I'gimentioning

confidence: 99%

“…Mixed oligonucleotide primers were designed from well-characterized partial amino acid sequences of C. syrnbiosum GDH (Fig. 1) [6]. Internal GDH sequences were used initially because the N-and C-terminal amino acid sequences were in doubt.…”

Section: Cloning Strategiesmentioning

confidence: 99%

The glutamate dehydrogenase gene of Clotridium symbiosum

Teller

Smith

McPherson

et al. 1992

European Journal of Biochemistry

Self Cite

105

101

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“…Differential gene loss, on the other hand, does not seem to have played an important role in the evolution of gdh genes in any of the three domains of life. Sequence comparisons for GDHs from a diverse range of sources show that the hexameric enzymes are similar whatever their coenzyme specificity [58,59]. On the other hand, the tetrameric enzymes are less well understood because of a lower number of characterized tetrameric GDHs.…”

Section: Classification Evolution and Structure Of Gdhsmentioning

confidence: 99%

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

Santero¹,

Hervás²,

Canosa³

et al. 2012

Dehydrogenases

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“…Sequence comparisons for GIuDH's from a diverse range of sources show that the hexameric enzymes are structurally homologous whatever their coenzyme specificity (Lilley et al, 1991;Baker et al, 1992). Specifically, the homology is poor for the Nterminal 50 residues, with homologous sequences being found for the next 350 residues.…”

Section: Introductionmentioning

confidence: 99%

Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophilePyrococcus furiosus

Yip

Baker²,

Britton³

et al. 1995

Acta Cryst D

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The NAD(P)-dependent glutamate dehydrogenase from Pyrococcus furiosus has been crystallized by the hanging-drop method of vapour diffusion using lithium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group P422~2 with unit-cell dimensions of a = b = 167.2, c = 172.9 A,. Consideration of the values of Vm and possible packing of the molecules within the cell suggest that the asymmetric unit contains a trimer. P. furiosus belongs to the family of Archaea and is one of the most thermostable organisms known, having an optimal growth temperature of 376 K. The glutamate dehydrogenase isolated from this organism has a half-life of 12 h at 373 K and, therefore, the determination of the structure of this enzyme will be important in advancing our understanding of how proteins are adapted to enable them to survive at such extreme temperatures.

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The partial amino acid sequence of the NAD -dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity

Cited by 39 publications

References 32 publications

The glutamate dehydrogenase gene of Clotridium symbiosum

The glutamate dehydrogenase gene of Clotridium symbiosum

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophilePyrococcus furiosus

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