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J. Biol. Chem., Vol. 275, Issue 50, 39529-39542, December 15, 2000
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From the Department Bioquímica y Biología
Molecular, Facultad de Veterinaria, Universidad de León, 24007 León, España and ¶ Los Álamos National
Laboratory, Biosciences Division,
Los Álamos, New Mexico 87545
A 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 The nucleotide sequence reported in this paper has been submitted
to the DDBJ/GenBankTM/EBI Data Bank with
accession number AF218569. This work is dedicated to Prof. J. R. Villanueva.
A New Class of Glutamate Dehydrogenases (GDH)
BIOCHEMICAL AND GENETIC CHARACTERIZATION OF THE FIRST MEMBER,
THE AMP-REQUIRING NAD-SPECIFIC GDH OF STREPTOMYCES
CLAVULIGERUS*
,
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 °C. Under these conditions, GDH
displayed hyperbolic behavior toward ammonia (Km,
33 mM) and sigmoidal responses to changes in
-ketoglutarate (S0.5 1.3 mM;
nH 1.50) and NADH (S0.5 20 µM; nH 1.52) concentrations.
Aspartate and asparagine were found to be allosteric activators. This
enzyme is inhibited by an excess of NADH or
NH4+, 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 Km 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.
*
This investigation was supported by Comisión
Interministerial de Ciencia y Tecnología, Madrid, Spain, Grant
AMB97-0603-C02-01, Fondo Europeo de Desarrollo Regional Grant
1FD97-0245, and Junta de Castilla y León Grant LE 42/96.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a fellowship from Comisión Interministerial de
Ciencia y Tecnología, Madrid, Spain.
§
Recipient of a fellowship from Fondo Europeo de Desarrollo.
To whom correspondence should be addressed: E-mail:
dbbjlr@unileon.es.
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