A Highly Active, Soluble Mutant of the Membrane-Associated (S)-Mandelate Dehydrogenase from Pseudomonas putida

Authors:
Publisher website
Google Scholar
Abstract
(S)-Mandelate dehydrogenase (MDH) from Pseudomonasputida, a member of the flavin mononucleotide-dependent α-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry32, 12959−12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH−GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH−GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, ∼39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH−GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH−GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.

This publication has 7 references indexed in Scilit: