A NOVEL STRUCTURAL BASIS FOR MEMBRANE ASSOCIATION OF A PROTEIN - CONSTRUCTION OF A CHIMERIC SOLUBLE MUTANT OF (S)-MANDELATE DEHYDROGENASE FROM PSEUDOMONAS-PUTIDA

The (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida (ATCC 12 633) is the only membrane-associated member of a homologous family of FMN-dependent, alpha-hydroxy acid dehydrogenases/oxidases that include s the structurally characterized glycolate oxidase from spinach (GOX). We have correlated the membrane association of MDH to a polypeptide s egment in the interior of the primary sequence. This has been accompli shed by construction of a chimeric enzyme in which the putative membra ne-binding segment in MDH has been deleted and replaced with the corre sponding 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 near ly the same when (S)-phenyllactate is used as substrate. To the best o f our knowledge, this is the first example of a membrane-associated pr otein in which an internal polypeptide segment anchors the protein to the membrane.