A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida.

Overview

The (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida (ATCC 12633) is the only membrane-associated member of a homologous family of FMN-dependent, alpha-hydroxy acid dehydrogenases/oxidases that includes the structurally characterized glycolate oxidase from spinach (GOX). We have correlated the membrane association of MDH to a polypeptide segment in the interior of the primary sequence. This has been accomplished by construction of a chimeric enzyme in which the putative membrane-binding segment in MDH has been deleted and replaced with the corresponding segment from the soluble GOX. The resulting chimera, MDH-GOX, is soluble and retains partial catalytic activity (approximately 1%) using (S)-mandelate as substrate. In contrast, the activities of both the membrane-associated wild-type MDH and the soluble MDH-GOX are nearly the same when (S)-phenyllactate is used as substrate. To the best of our knowledge, this is the first example of a membrane-associated protein in which an internal polypeptide segment anchors the protein to the membrane.