REDESIGN OF SACCHAROMYCES-CEREVISIAE FLAVOCYTOCHROME B(2) - INTRODUCTION OF L-MANDELATE DEHYDROGENASE-ACTIVITY

Flavocytochrome b(2) from Saccharomyces cerevisiae is an L-lactate deh ydrogenase which exhibits only barely detectable activity levels towar ds another 2-hydroxyacid, L-mandelate. Using protein engineering metho ds we have altered the active site of flavocytochrome b(2) and success fully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on th e ability of the enzyme to utilize L-mandelate as a substrate. The dou ble 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), whi ch 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-ra y structure of S. cereoisine flavocytochrome b(2) led us to conclude t hat 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, t he activity with L-mandelate of the independently expressed flavodehyd rogenase domain (FDH), was examined and found to be higher than that s een with the wildtype enzyme. In addition, the double mutation of Ala- 198 --> Gly and Leu-230 --> Ala introduced into FDH produced the great est mandelate dehydrogenase activity increase, with a k(cat) value mor e 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 bet ter mandelate dehydrogenase than a lactate dehydrogenase.