Flavocytochrome b2 from Saccharomyces cerevisiaeis 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 b2 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 kcat value (25 °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 kcat/Km values) that is 6-fold higher with l-mandelate than it is with l-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 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 wild-type 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 kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) 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.

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