RESUMO
Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis. Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Piridoxal Quinase/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Catálise , Cátions Bivalentes/farmacologia , Clonagem Molecular , Escherichia coli/genética , Flores/enzimologia , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Germinação/genética , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Caules de Planta/enzimologia , Caules de Planta/genética , Piridoxal Quinase/efeitos dos fármacos , Piridoxal Quinase/isolamento & purificação , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/biossíntese , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/genética , Sementes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Asthmatics treated with theophylline, a potent inhibitor of pyridoxal kinase and therefore a vitamin B6 antagonist, demonstrated a significant correlation (r = 0.71; p < 0.001) between drug plasma levels and erythrocyte pyridoxal kinase activities. A cross-over, placebo controlled study was completed on 15 healthy volunteers to investigate the mechanism by which theophylline induces pyridoxal kinase activity. The subjects were supplemented with vitamin B6 or placebo for two weeks before theophylline therapy was started. Vitamin B6 supplementation resulted in a four-fold increase in circulating pyridoxal 5'-phosphate levels, while placebo had no effect. When theophylline therapy was commenced, erythrocyte pyridoxal kinase activities increased significantly (p < 0.001) irrespective of whether vitamin B6 or placebo was supplemented. It is concluded that a depressed vitamin B6 status is not responsible for higher erythrocyte pyridoxal kinase activities encountered during theophylline therapy, but that the drug is directly responsible for elevated enzyme levels.
