The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli.
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(r) 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-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-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 site-directed mutagenesis.
Teller
J K
JK
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, England.
Smith
R J
RJ
McPherson
M J
MJ
Engel
P C
PC
Guest
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase