Enolase from the ectomycorrhizal fungus Tuber borchii Vittad.: biochemical characterization, molecular cloning, and localization.
Enolase from Tuber borchii mycelium was purified to electrophoretical homogeneity using an anion-exchange and a gel permeation chromatography. Furthermore, the corresponding gene (eno-1) was cloned and characterized. The purified enzyme showed a higher affinity for 2-PGA (0.26 mM) with respect to PEP; the stability and activity of enolase were dependent of the divalent cation Mg2+. T. borchii eno-1 has an ORF of 1323 bp coding for a putative protein of 440 amino acids and Southern blotting analysis revealed that the gene is present as a single copy in T. borchii. The enzymatic activity and the mRNA expression level evaluated in mycelia grown either in different carbon sources, in pyruvate or during starvation were the same in all the conditions tested, while biochemical and Northern blotting analyses performed with mycelia at different days of growth showed T. borchii eno-1 regulation in response to the growth phase. Finally, Western blotting analysis demonstrated that enolase is localized only in the cytosolic fraction confirming its important role in glycolysis.
Polidori
Emanuela
E
Istituto di Chimica Biologica Giorgio Fornaini, Università degli Studi di Urbino Carlo Bo, Via A. Saffi, 2, 61029 Urbino (PU), Italy.
Saltarelli
Roberta
R
Ceccaroli
Paola
P
Buffalini
Michele
M
Pierleoni
Raffaella
R
Palma
Francesco
F
Bonfante
Paola
P
Stocchi
Vilberto
V
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D005988:Glyceric Acids / Q000378:metabolism; D007438:Introns / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D011401:Promoter Regions, Genetic; D039104:RNA 3' Polyadenylation Signals / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000502:physiology; D024363:Transcription Initiation Site
D003067:Coenzymes; D004271:DNA, Fungal; D005988:Glyceric Acids; C008885:2-phosphoglycerate; D010728:Phosphoenolpyruvate; D010751:Phosphopyruvate Hydratase; D008274:Magnesium