L-mandelate dehydrogenase activity
نویسندگان
چکیده
Flavocytochrome b # from Saccharomyces cereisiae is an lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, -mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b # 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 -mandelate as a substrate. The doublemutation of Ala-198!Gly and Leu-230!Ala results in an enzyme with a k cat value (25 °C) with -mandelate of 8.5 s−", 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 -mandelate than it is with -lactate. Closer examination of the X-ray structure of S. cereisiae flavocytochrome b # led us to
منابع مشابه
An assay procedure for the vitamin K1 2,3-epoxide-reducing system of rat liver involving high-performance liquid chromatography.
since measurements of O2 uptake with intact bacteriaor extracts indicated approximately the same ratio of activities. Further, bacteria growing on D(-)-mandelate did not accumulate benzaldehyde, which shows that D(-)-mandelate dehydrogenase, rather than benzaldehyde dehydrogenase I (Cook et al., 1975), was now rate-limiting. D(-)-Mandelate dehydrogenase resembles L(+)-mandelate dehydrogenase in...
متن کاملMembrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties.
Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes ...
متن کاملInitial reactions involved in the dissimilation of mandelate by Rhodotorula graminis.
Rhodotorula graminis utilized DL-mandelate, L(+)-mandelate, and D(-)-mandelate as sole sources of carbon and energy. Growth on these aromatic substrates resulted in the induction of an NAD-dependent D(-)-mandelate dehydrogenase and a dye-linked L(+)-mandelate dehydrogenase, each catalyzing the stereospecific conversion of its respective enantiomer of mandelate to benzoylformate. Benzoylformate ...
متن کاملRegulation of the mandelate pathway in Pseudomonas aeruginosa.
The pathway of mandelate metabolism in Pseudomonas aeruginosa is composed of the following steps: l(+)-mandelate --> benzoylformate --> benzaldehyde --> benzoate. These three steps are unique to mandelate oxidation; the benzoate formed is further metabolized via the beta-ketoadipate pathway. The first enzyme, l(+)-mandelate dehydrogenase, is induced by its substrate. The second and third enzyme...
متن کاملIdentification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633.
The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarb...
متن کامل