Biochemical and molecular characterization of NADP-glutamate dehydrogenase from the ectomycorrhizal fungus Tuber borchii

Abstract

• NADP-glutamate dehydrogenase (NADP-GDH) from Tuber borchii was purified and the corresponding gene was cloned in order to elucidate the physiological role of the enzyme in this ectomycorrhizal fungus. • NADP-GDH was purified using an anion-exchange column followed by affinity chromatography. The complete gene was cloned from a 30-d-old-mycelium cDNA library and characterized. • T. borchii NADP-GDH appears to be physically and kinetically similar to those from other fungi and the deduced amino acid sequence of the gdh gene showed a significant similarity to other fungal NADP-dependent GDHs. Biochemical and Northern blotting analyses carried out with mycelia grown on different nitrogen sources clearly showed that the regulation of T. borchii NADP-GDH in response to different nitrogen sources was markedly different from the responses of the NADP-GDHs of other ascomycetes. Northern blotting analyses highlighted that the gdh gene was also expressed in the symbiotic phase. • The biochemical and molecular data suggest that the fungal NADP-GDH contributes to the primary nitrogen metabolism in the ectomycorrhizal tissues.

Keywords: Tuber borchii; expression analysis; gene cloning; glutamate dehydrogenase; purification.