Characterization of Trypanosoma brucei pyridoxal kinase: purification, gene isolation and expression in Escherichia coli.
Pyridoxal kinase catalyzes the ATP-dependent phosphorylation of vitamin B6, generating pyridoxal-5'-phosphate, an important cofactor for many enzymatic reactions. Pyridoxal kinase was purified 4300-fold to homogeneity from Trypanosoma brucei and peptides generated by proteolysis were subjected to amino acid sequence analysis. The peptide sequence information was used to generate a partial clone of T. brucei pyridoxal kinase by polymerase chain reaction (PCR), which in turn was used to screen a T. brucei genomic library for a full length clone. The 903-bp gene was sequenced and found to encode a 300-amino acid protein. The deduced amino acid sequence contains all of the peptide sequences obtained from the proteolytic cleavage of the native enzyme and shares 28% sequence identity with a putative Escherichia coli pyridoxal kinase, identified for its ability to compliment pyridoxal kinase deficient cells. The T. brucei pyridoxal kinase gene was expressed in E. coli and the purified enzyme was found to have pyridoxal kinase activity, confirming that this gene encodes the functional T. brucei enzyme. Native and recombinant pyridoxal kinase have a monomer molecular weight of 37 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are dimers in solution. Native T. brucei pyridoxal kinase catalyzes the phosphorylation of pyridoxal with a specific activity of 990 nmol min(-1) per mg and apparent Km values for pyridoxal and ATP of 22 and 9 microM. respectively. Substrate inhibition is observed for pyridoxal. Similar results were obtained for the recombinant enzyme.
Scott
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TC
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Phillips
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MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011731:Pyridoxal Kinase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D011731:Pyridoxal Kinase