Cloning and characterization of Arabidopsis thaliana pyridoxal kinase.
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.
Lum
Hon-Kei
HK
Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Hong Kong Special Administrative Region, People's Republic of China.
Kwok
Francis
F
Lo
Samuel C L
SC
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D035264:Flowers / Q000201:enzymology / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D018525:Germination / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D018547:Plant Stems / Q000201:enzymology / Q000235:genetics; D011731:Pyridoxal Kinase / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D011731:Pyridoxal Kinase