RESUMO
Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought.
Assuntos
Arabidopsis/enzimologia , L-Iditol 2-Desidrogenase/metabolismo , Sorbitol/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomassa , Citosol/enzimologia , Desidratação , Flores/enzimologia , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Cinética , L-Iditol 2-Desidrogenase/genética , Dados de Sequência Molecular , Mutação , NAD/metabolismo , Especificidade de Órgãos , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/ultraestrutura , Caules de Planta/enzimologia , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/ultraestrutura , Proteínas Recombinantes de Fusão , Ribitol/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Sementes/ultraestrutura , Alinhamento de Sequência , Xilitol/metabolismoRESUMO
A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 --> Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 --> V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 --> P196 mutation that would disrupt a surface alpha-helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.
Assuntos
Mutação , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , D-Xilulose Redutase , Klebsiella pneumoniae/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , Estrutura Secundária de Proteína , Ribitol/metabolismo , Alinhamento de Sequência , Análise de Sequência , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/genética , Xilitol/metabolismoRESUMO
Pullularia pullulans grown on D-xylose induces the synthesis of a xylitol dehydrogenase capable of oxidizing xylitol to D-xylulose in the presence of NAD according to the following reaction: Xylitol + NAD+ in equilibrium to D-xylulose + NADH + H+. Cells grown on D-glucose do not show appreciably xylitol dehydrogenase activity. However, synthesis of the enzyme begins when those cells were transferred to a liquid medium containing D-xylose. This evidence suggests that xylitol dehydrogenase is an induced enzyme. The purified xylitol dehydrogenase of Pullularia pullulans is quite specific for xylitol, unlike similar dehydrogenases from other sources, which attack a variety of polyols of variable chain length. This property makes the enzyme useful for the determination of xylitol, even in the presence of other polyols.