Abstract
Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.
Highlights
Supplementary key words sterol biosynthesis sterol biosynthetic enzymes inhibition suicide substrates crystal structure cytochrome P450 Trypanosoma brucei Trypanosoma cruzi
Responses of protozoan sterol 14␣-demethylase (CYP51) to substrate addition are significantly more generous, ranging from 35% for the T. cruzi ortholog [23] to 50% and even to 90% for the enzymes from T. brucei [21] and L. infantum [22], respectively (C29norlanosterol)
Upon concentration required initial rate of catalysis (5 min reaction, ف50% substrate conversion without an inhibitor) and long-term inhibition (1 h reaction, 100% substrate conversion without an inhibitor) were monitored (Table 1). 3H-Radiolabeled obtusifoliol was used as a substrate with T. brucei, L. infantum, and the I105F mutant of T. cruzi CYP51, and 3H-eburicol (24-methylenedihydrolanosterol) was used as a substrate with T. cruzi CYP51
Summary
CYP51 proteins and their electron donor partner (T. brucei cytochrome P450 reductase) were purified and assayed as previously described [21,22,23]. The desired product (~30 mg) was obtained by removal of the C3-protecting group by adding anhydrous diethyl ether (20 ml) followed by addition of lithium aluminum hydride (4 mg) under a stream of nitrogen. The standard reaction mixture contained 1 μM CYP51 and 2 μM cytochrome P450 reductase, 50 M radiolabeled sterol substrate, and varying concentrations of MCP. The ability of CYP51 samples complexed with MCP to be chemically reduced by sodium dithionite and to form complexes with carbon monoxide was confirmed by CO difference spectra. To compare effects of MCP on the enzymatically reduced heme moiety of T. brucei versus T. cruzi CYP51s, CO difference spectra were monitored after 10 min CYP51 incubation in the reconstituted enzyme reaction mixture either with their substrates or with MCP
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