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UDP-apiose/UDP-xylose synthase. Subunit composition and binding studies.

U Matern, H Grisebach

    European Journal of Biochemistry
    |April 1, 1977
    PubMed
    Summary
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    This study purified UDP-apiose/UDP-xylose synthase, revealing it comprises two proteins. The larger protein possesses enzymatic activity, while the smaller enhances stability, crucial for apiose synthesis.

    Area of Science:

    • Biochemistry
    • Enzymology
    • Plant Science

    Background:

    • Parsley cell suspension cultures produce UDP-apiose/UDP-xylose synthase.
    • This enzyme synthesizes UDP-apiose and UDP-xylose from UDP-D-glucuronic acid (UDP-GlcUA).
    • Understanding enzyme composition is key to elucidating biosynthetic pathways.

    Purpose of the Study:

    • To purify and characterize the UDP-apiose/UDP-xylose synthase from parsley.
    • To determine the subunit composition and functional roles of the enzyme's protein components.
    • To investigate the substrate binding and catalytic mechanism of the enzyme.

    Main Methods:

    • 1400-fold purification of the enzyme using an improved method.
    • Dodecylsulfate-gel electrophoresis and sedimentation equilibrium measurements for molecular weight determination.

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  • Chromatographic techniques (DEAE-cellulose, omega-aminoalkyl-Sepharose, gel filtration) and crosslinking assays.

    • Main Results:

    • The purified enzyme consists of two proteins (65,000 and 86,000 Mr) in a 1:0.7-1:0.9 molar ratio.
    • Separation was achieved using DEAE-cellulose in urea; the 86,000 Mr protein retained activity, while the 65,000 Mr protein was essential for stability.
    • The 86,000 Mr protein binds 0.5 mol UDP-GlcUA/mol and 0.5 mol NAD+/mol in the presence of UDP, with no evidence of Schiff base intermediates.

    Conclusions:

    • UDP-apiose/UDP-xylose synthase is a heterodimer crucial for apiose biosynthesis in parsley.
    • The 86,000 Mr protein is the catalytic subunit, while the 65,000 Mr protein is a stability factor.
    • The enzyme does not appear to utilize a Schiff base mechanism for the 4-keto intermediate in apiose synthesis.