Metabolism of [C-14] glutamate and [C-14] glutamine by the ectomycorrhizal fungus Paxillus involutus

Abstract:
To examine pathways of glutamate and glutamine metabolism in the ectomycorrhizal fungus Paxillus involutus, tracer kinetic experiments were performed using L-[U-C-14]glutamate and L-[U-C-14]glutamine and the enzyme inhibitors methionine sulfoximine (MSX), azaserine (AZA) and aminooxyacetate (AOA). When [C-14]glutamate was supplied to fungal cultures, 25% of the radioactivity of the amino acid fraction was incorporated into glutamine after 5 min feeding, but MSX inhibited incorporation of C-14 into glutamine by 85%, suggesting the rapid operation of glutamine synthetase. Conversely, when P. involutus was fed with [C-14]glutamine, 46% of the label was found in glutamate within 30 min of feeding and AZA inhibited glutamate formation by 90%. Taken together, these data indicate that glutamate synthase (GOGAT) is the major enzyme of glutamine degradation. In addition, the strong inhibition of glutamine utilization by AOA indicates that glutamine catabolism in P. involutus might involve a transamination process as an alternative pathway to GOCAT for glutamine degradation. The high (CO2)-C-14 evolution shows that glutamate and glutamine are further actively consumed as respiratory substrates, being channelled through the tricarboxylic acid (TCA) cycle and oxidized as CO2. It appears that synthesis of amino acid precursors during TCA cycle operation is an essential step far aspartate and alanine synthesis through aminotransferase activities in P. involutus.