TAILIEUCHUNG - Báo cáo khoa học: The metabolic role and evolution of L-arabinitol 4-dehydrogenase of Hypocrea jecorina

L-Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungusHypocrea jecorina(anamorph: Trichoderma reesei) has been implicated in the catabolism ofL-arabinose, and genetic evidence also shows that it is involved in the catabolism of D-xylose in xylitol dehydrogenase (xdh1) mutants and of D-galactose in galactokinase (gal1) mutants of H. order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme inEscherichia coliand purified it to physical homogeneity | Eur. J. Biochem. 271 1864-1872 2004 FEBS 2004 doi The metabolic role and evolution of L-arabinitol 4-dehydrogenase of Hypocrea jecorina Manuela Pail1 Thomas Peterbauer2 Bernhard Seiboth1 Christian Hametner3 Irina Druzhinina1 and Christian P. Kubicek1 1 Division of Gene Technology and Applied Biochemistry Institute of Chemical Engineering TU Wien Vienna 2Institute of Ecology University of Vienna 3Institute of Applied Synthetic Chemistry TU Wien Vienna Austria L-Arabinitol 4-dehydrogenase Lad1 of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina anamorph Trichoderma reesei has been implicated in the catabolism of L-arabinose and genetic evidence also shows that it is involved in the catabolism of D-xylose in xylitol dehydrogenase xdh1 mutants and of D-galactose in galactokinase gal1 mutants of H. jecorina. In order to identify the substrate specificity of Lad1 we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols L-arabini-tol and hexitols D-allitol D-sorbitol L-iditol L-mannitol to the same corresponding ketoses as mammalian sorbitol dehydrogenase SDH albeit with different catalytic efficacies showing highest kcat Km for L-arabinitol. However it oxidized galactitol and D-talitol at C4 exclusively yielding L-xylo-3-hexulose and D-arabino-3-hexulose respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for D-sorbitol. A lad1 gene disruptant was almost unable to grow on L-arabinose grewextremely weakly on L-arabinitol D-talitol and galactitol showed reduced growth on D-sorbitol and D-galactose and a slightly reduced growth on D-glucose. The

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