plant surface microbiology.pdf

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A. Javelle et al.
Glu. Glutamine synthetase (GS, EC 6.3.1.2) incorporates ammonium into the
carboxyl group of Glu to form Gln. In turn, the Glu and Gln formed serve as
donors in transamination and amido nitrogen transfer reactions. Glu is an
essential amino N donor for many transaminases and Gln amide nitrogen is
used to synthesize many essential metabolites, such as nucleic acids, amino
sugars, His, Tyr, Asn, and various cofactors. Both Glu and Gln are essential for
protein synthesis. Glutamate synthase (GOGAT) is responsible for the reductive
transfer of amide N to a-ketoglutarate for the generation of two molecules
of glutamate, one of which is recycled for glutamine biosynthesis. The
net result of the combined action of GS and GOGAT is the synthesis of glutamate
from ammonium and a-ketoglutarate, frequently referred to as the
GS/GOGAT cycle.
6.1 Role and Properties of Glutamate Dehydrogenase
Most of the ascomycete and basidiomycete fungi possess two glutamate dehydrogenases
(GDH), each specific for one of the two cofactors. A catabolic role
has been assigned to the NAD-specific enzyme (EC 1.4.1.2), whereas the
NADP-specific enzyme (EC 1.4.1.4) has been involved in glutamate biosynthesis
(Ferguson and Sims 1971). This was confirmed in the ectomycorrhizal
fungus Laccaria laccata where both enzymes were purified and characterized
(Brun et al. 1992; Botton and Chalot 1995; Garnier et al. 1997). Both enzymes
revealed biphasic kinetics with two different Km values for glutamate, the
NADP-GDH exhibiting a positive cooperativity, and the NAD-GDH a negative
cooperativity. At all tested concentrations of glutamate, NAD-GDH showed a
higher affinity for this amino acid than the NADP-specific enzyme. This was
especially true at low glutamate concentrations where the affinity of NADP-
GDH was very low (Km value: 100 mM), while the affinity of NAD-GDH was
maximal (Km value: 0.24 mM). In addition, NADP-GDH was found to have a
considerably higher affinity for ammonium than the NAD-dependent
enzyme and was not calcium-dependent for its activity, contrary to what was
found with the latter enzyme. The native NADP-GDHs purified from Cenococcum
geophilum (Martin et al. 1983), and Laccaria bicolor (Ahmad and
Hellebust 1991), revealed properties roughly similar to those of the Laccaria
laccata NADP-GDH.
Activities of glutamate dehydrogenase in conjunction with glutamine synthetase
in the free-living Pezizella ericae, Cenococcum geophilum (Martin et
al. 1983), and Laccaria laccata (Lorillou et al. 1996), were found to be high and
sufficient to sustain high rates of nitrogen assimilation. In cultured Cenococcum
geophilum,NH 4 + is assimilated via the glutamate dehydrogenase pathway
and the glutamate formed is rapidly used to synthesize glutamine. Ammonium
ion assimilation leads to the synthesis of large amounts of glutamine,
alanine and arginine (Martin et al. 1987). These amino acids represent the