Re-design of Saccharomyces cerevisiae flavocytochrome b(2): introduction of L-mandelate dehydrogenase activity

Rhona Sinclair, Graeme A Reid, Stephen K Chapman

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    Abstract

    Flavocytochrome b(2) from Saccharomyces cerevisiae is an L-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, L-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b(2) and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize L-mandelate as a substrate. The double mutation of Ala-198 -->Gly and Leu-230 -->Ala results in an enzyme with a k(cat) value (25 degrees C) with L-mandelate of 8.5 s(-1), which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by k(cat)/K-m values) that is 6-fold higher with L-mandelate than it is with L-lactate. Closer examination of the X-ray structure of S. cereoisine flavocytochrome b(2) led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme. To test this idea, the activity with L-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wildtype enzyme. In addition, the double mutation of Ala-198 --> Gly and Leu-230 --> Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a k(cat) value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (k(cat)/K-m) of this mutant enzyme was more than 20-fold greater with L-mandelate than with L-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.

    Original languageEnglish
    Pages (from-to)117-120
    Number of pages4
    JournalBiochemical Journal
    Volume333
    Issue number1
    Publication statusPublished - 1 Jul 1998

    Keywords

    • SITE
    • ELECTRON-TRANSFER
    • B2
    • FLAVIN
    • YEAST FLAVOCYTOCHROME-B2
    • L-LACTATE DEHYDROGENASE

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