Catabolism of α-Ketoglutarate by a sucA Mutant of Bradyrhizobium japonicum : Evidence for an Alternative Tricarboxylic Acid Cycle

Abstract

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing ␣-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of ␣-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate ␣-[U-14 C]ketoglutarate and [U-14 C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for ␣-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-14 C]glutamate very slowly, the ␥-aminobutyrate shunt is unlikely to be the pathway responsible for ␣-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent ␣-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP i . Thin-layer chromatography showed that the product of ␣-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent ␣-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for ␣-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.

Variant tricarboxylic acid cycle in Mycobacterium tuberculosis : Identification of α-ketoglutarate decarboxylase

“…To our knowledge, throughout species, only three Kgd activities have been reported: in Euglena gracilis (34), in B. japonicum (17) and in association with a bifunctional protein cloned from E. coli, MenD (35). The activities in Euglena gracilis and the sucA mutant strain of B. japonicum have not been ascribed to specific genes or proteins, so it is not possible to tell whether the enzymes involved are homologs of Mtb's Kgd.…”

“…We were able to detect high levels of glutamate dehydrogenase activity in M. smegmatis but not in Mtb or M. bovis bacillus Calmette-Guérin (data not shown). In Euglena gracilis (34) and rhizobia such as sucA B. japonicum, Mesorhizobium loti, and Rhizobium leguminosarum, it was suggested that the bypass pathway from ␣-ketoglutarate to succinate via SSA represents an adaptation to microaerophilic conditions inside legume nodules (17). Similarly the Kgd-GabD1 bypass pathway in Mtb may help the pathogen cope with conditions in host cells that are genuinely hypoxic, or, as appears to be the case in Mtb residing in macrophages in vitro, misperceived as hypoxic, perhaps because Mtb's oxygen-sensing mechanisms have been damaged by host-derived nitric oxide (3).…”

“…We were not able to detect pyruvate, citrate, aconitate, isocitrate, ␣-ketoglutarate, succinyl-CoA, and SSA under our conditions, which may reflect the poor stability and low recovery of their authentic standards (Table 3). The enzyme activities from Mtb, M. bovis BCG, and M. smegmatis compared to that published for B. japonicum (17) and potato SSADH (23). NR, not reported; NM, not measured.…”

Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis

“…However, due to interference from the crude cell extracts at 240 nm, the formation of fumarate was monitored by following the increase in absorbance at 300 nm. 2-Oxoglutarate dehydrogenase assay was performed by monitoring the release of 14 CO 2 from [1-14 C]2-oxoglutarate in the presence of NAD + and Coenzyme A (Green et al, 2000). For all assays, a milliunit (mU) of activity is defined as 1 nmol substrate reacted min 21 .…”

Euglena gracilis Rhodoquinone:Ubiquinone Ratio and Mitochondrial Proteome Differ under Aerobic and Anaerobic Conditions

“…The resulting acetyl-CoA enters a modified citric acid cycle, which entails a shunt via succinate semialdehyde as in the ␣-proteobacterium Bradyrhizobium (18), circumventing the step catalyzed by ␣-ketoglutarate dehydrogenase. Under anaerobic conditions, pyruvate:NADPϩ oxidoreductase constitutes the key enzyme for a unique wax ester fermentation.…”