Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus

Abstract

The gene for the catabolic NAD-linked glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was cloned by selection in Escherichia coli for complementation of a biosynthetic defect. Cloned fragments containing the gene and the P. asaccharolyticus transcription and translation signals are very highly expressed in E. coli. The nucleotide sequence of the cloned gene was determined. It codes for a polypeptide of 421 amino acids, the sequence of which is similar to those of the NADP-accepting glutamate dehydrogenases. The sequence similarity of this protein to the mammalian glutamate dehydrogenases, which accept both NADP and NAD, is greater than its similarity to the bacterial NADP-specific dehydrogenases, suggesting that this NAD-specific bacterial glutamate dehydrogenase and the NADP-specific bacterial dehydrogenases diverged separately from the line leading to the dual-specificity mammalian glutamate dehydrogenases.As part of a program to develop live cell catalysts for the manufacture of amino acids by transatination of the corresponding alpha-keto acids, we cloned in Escherichia coli the gene for the NAD-dependent glutamate dehydrogenase of an anaerobic gram-positive coccus, Peptostreptococcus asaccharolyticus. This glutamate dehydrogenase is one of the few bacterial NAD-dependent glutamate dehydrogenases that have been purified and characterized (14,16,18,33). In P. asaccharolyticus, it acts in the hydroxyglutarate fermentation of glutamate, in which its normal role is the degradation of glutamate (15). We selected for the reverse of this reaction to obtain the clone. In this paper, we report the isolation, expression, and nucleotide sequence analysis of the P. asaccharolyticus gene for glutamate dehydrogenase. We compare the amino acid sequence of the translated gene with those of other glutamate dehydrogenases to show that this glutamate dehydrogenase is more closely related to the dual-specificity (NAD-and NADP-accepting) mammalian glutamate dehydrogenases than to the NADP-linked glutamate dehydrogenase of E. coli. MATERIALS AND METHODSBacterial strains. The strains used in this work are listed in Table 1. To construct strain Q100, we moved a host restriction mutation into a glutamate auxotroph by cotransduction with a nearby TnJO insertion. Strain PA340 was transduced with P1 phage prepared from strain LCK8, and the Tetr transductants were scored for the retention of the hsdR2 locus by comparing the efficiency of plaque formation when host-modified and unmodified P1 phages were plated on each transductant. Selection for the loss of the TnJO insertion was carried out by the method of Bochner and coworkers (2). DNA isolation. P. asaccharolyticus cells were grown anaerobically in Difco thiol broth. Cycloserine (6 ,ug/ml) was added just before the cells entered stationary phase in order to make them more susceptible to lysozyme action. The harvested cells were washed in 0.15 M NaCl and were then * Corresponding author. resuspended in 18.75% sucrose-37.5 mM Tris-HCl buffer (pH 8) containing 5 mg of lys...

The glutamate dehydrogenase gene of Clotridium symbiosum

“…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.…”

“…The subunits of eubacterial and fungal GDHs are smaller than their animal counterparts and sequence comparisons have shown that they are more closely related [16,401. Nevertheless, it is clear that similar folding patterns are achieved by relatively different sequences.…”

“…Recombinant plasmid pG1, a pBR322 derivative carrying the Peptostreptococcus asaccharolyticus gdh gene, was a gift from Dr. B. Snedecor [16]. C. symbiosum DNA libraries were constructed in AEMBL3, AongC and Agtl0 (Stratagene) according to the manufacturers' instructions.…”

“…These included screening libraries of C. symhiosum DNA cloned in AEMBL3, RongC and igtlO. Antibodies raised against C. syrnbiosum GDH, mixed oligonucleotides encoding amino acid sequences deduced from G D H peptides, and fragments of both the E. coli [29] and Peptostreptococcus asaccharolyticus gdh [16] genes, were all used as probes to screen these libraries. No gdh-containing plaques were detected either before or after amplification of the in-vituo-packaged phage libraries.…”

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

“…NAD ϩ -dependent GDHs are either tetrameric or hexameric and are mainly involved in glutamate catabolism, while NADP ϩ -dependent GDHs are hexameric and are mainly involved in ammonia assimilation and hence in glutamate synthesis. Catabolic NAD ϩ -dependent GDHs have been found in several microorganisms, including Saccharomyces cerevisiae (34), Candida utilis (9), Neurospora crassa (2,8), Streptomyces fradiae (17), Peptostreptococcus asaccharolyticus (28), Halobacterium halobium (3), Thermus thermophilus (23), Clostridium symbiosum (32), and Clostridium difficile (16), but only a few of these enzymes have been genetically and biochemically characterized (the C. symbiosum, C. difficile, and P. asaccharolyticus enzymes). Of the known gdh genes, the gdh gene of P. asaccharolyticus seems to be suitable for expression in L. lactis, since it is a gene from a gram-positive coccus that is phylogenetically closely related to L. lactis.…”