Defects in vesicle core induced by Escherichia coli DHODH

¹ Universidade de São Paulo

^* To whom correspondence should be addressed. E-mail: ajcosta{at}if.sc.usp.br.

Submitted on August 17, 2007
Revised on September 18, 2007
Accepted on 10 October 2007

	Abstract

Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. E. coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used Electronic Spin Resonance to study the interaction of EcDHODH with vesicles of DOPC/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 10-PC in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.

Key Words: N-terminal extension, defects, dihydroorotate dehydrogenase, electron spin resonance, vesicles