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Title

A new class of glutamate dehydrogenases (GDH). Biochemical and genetic characterization of the first member, the AMP-requiring NAD-specific GDH of Streptomyces clavuligerus

AuthorsMiñambres Rodríguez, Baltasar; Olivera, Elías R.; Jensen, Roy A.; Luengo, José M.
KeywordsBeta-lactam
Cephalosporin production
Pyrococcus-furiosus
Sequence alignment
Clavulanic acid
Purification
Enzyme
Evolution
Synthetase
Mechanism
Issue Date2000
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry 275(50): 39529-39542 (2000)
AbstractA new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an alpha(6) oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mm phosphate buffer, pH 7.0, at 30 degrees C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K(m), 33 mm) and sigmoidal responses to changes in alpha-ketoglutarate (S(0.5) 1.3 mm; n(H) 1.50) and NADH (S(0.5) 20 microm; n(H) 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH(4)(+), by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K(m) for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed.
Description14 pages, 10 figures, 6 tables.-- PMID: 10924516 [PubMed].
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AF218569.
Publisher version (URL)http://dx.doi.org/10.1074/jbc.M005136200
URIhttp://hdl.handle.net/10261/12218
DOI10.1074/jbc.M005136200
ISSN0021-9258
Appears in Collections:(IGM) Artículos

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