Defects in vesicle core induced by escherichia coli dihydroorotate dehydrogenase

Abstract

Dihydroorotate dehydrogenase (DHODH) 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. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/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-phosphatidylcholine 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.

Figure 1

ESR spectra of spin labels (a) DPPTC, (b) 5-, (c) 10-, (d) 12-, and (e) 16-PC incorporated into vesicles of DOPC/Triton X-100 in the absence (solid line) and in the presence of EcDHODH (dashed line). Experimental conditions: microwave frequency 9.5 GHz; modulation amplitude 1.0 G; modulation frequency 100 kHz; microwave power 10 mW.

Figure 2

Chemical structures of the (a) acyl-chain (16-PC) and (b) headgroup (DPPTC) spin labels showing the principal magnetic axes (x_m, y_m, z_m).

Figure 3

Experimental (dashed line) and simulated (solid line) ESR spectra from (a) DPPTC, (b) 12-, and (c) 16-PC labels in mixtures of DOPC/Triton X-100 (left column) and DOPC/Triton X-100/EcDHODH (right column).

Figure 4

Experimental (dashed line) and calculated (solid line) ESR spectra from 5-PC spin probe in mixtures of (a) DOPC/Triton X-100 and (b) DOPC/Triton X-100/EcDHODH. b shows the individual components: 1 (bulk lipid, dotted line) and 2 (boundary lipid, dash-dotted line) obtained by means of NLSL simulations.

Figure 5

Experimental (dashed line) and calculated (solid line) ESR spectra from 10-PC spin probe in mixtures of (a) DOPC/Triton X-100 and (b) DOPC/Triton X-100/EcDHODH. b shows the individual components: 1 (bulk lipid, dotted line) and 2 (boundary lipid, dash-dotted line) obtained as described in the text.

Figure 6

Ribbon representation of EcDHODH structure emphasizing the hydrophobicity pattern for N-terminal domain (comprising residues in the two α-helices and one 3₁₀ helix) as determined by ProtScale software. The dehydrogenase-active domain is shown in cyan. Hydrophobic and hydrophilic residues in the N-terminal domain are colored in dark and light gray, respectively.