On the Two Components of Pyridoxal 5′-Phosphate Synthase from Bacillus subtilis

Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted...

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Bibliographic Details
Published in:The Journal of biological chemistry 2005-09, Vol.280 (37), p.32291-32300
Main Authors: Raschle, Thomas, Amrhein, Nikolaus, Fitzpatrick, Teresa B.
Format: Article
Language:English
Subjects:
Antioxidants - chemistry
Antioxidants - pharmacology
Bacillus subtilis
Bacillus subtilis - metabolism
Binding Sites
Carbon-Nitrogen Lyases - biosynthesis
Carbon-Nitrogen Lyases - chemistry
Chromatography
Chromatography, Gel
Chromatography, High Pressure Liquid
Cloning, Molecular
Cysteine - chemistry
DNA - chemistry
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Escherichia coli - metabolism
Escherichia coli Proteins - metabolism
Glutaminase - biosynthesis
Glutaminase - chemistry
Glutamine - chemistry
Immunochemistry
Isoxazoles - chemistry
Kinetics
Mass Spectrometry
Models, Chemical
Organophosphates - chemistry
Oxidoreductases - metabolism
Pentosephosphates - chemistry
Protein Structure, Tertiary
Pyridoxal Phosphate - chemistry
Quaternary Ammonium Compounds - chemistry
Spectrometry, Mass, Electrospray Ionization
Spectrophotometry
Threonine - analogs & derivatives
Threonine - chemistry
Time Factors
Ultraviolet Rays
Vitamin B 6 - chemistry
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Summary:Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-d -xylulose 5-phosphate and 4-phosphohydroxy-l-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5′-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M501356200