Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14α-Demethylase (CYP51) from Leishmania infantum

Abstract

Leishmaniasis is a major health problem that affects populations of ϳ90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/ function characterization of sterol 14␣-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14␣-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V max of ϳ10 and 8 min ؊1 , respectively), it is also found to 14␣-demethylate C4-dimethylated lanosterol (V max ‫؍‬ 0.9 min ؊1 ) and C4-des- These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.

Sterols in spermatogenesis and sperm maturation

“…Mammalian CYP51 from different species displays over 90% sequence identity at the protein level ( 15 ). Moreover, CYP51 from distant eukaryotic organisms has been found to be strikingly similar at the structural level (16)(17)(18)(19). These data indicate that CYP51 function has remained highly conserved throughout evolution, suggesting an essential role of this gene in organisms of various taxa.…”

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

“…MCP was added to the sample cuvette in the concentration range 0.25-5.0 µM from a 1 mM stock solution in 45% hydroxypropyl-␤ -cyclodextrin (Cyclodex). The spin state of the P450 samples was estimated from the absolute absorbance spectra ( 22 ). The percentage of low-to-high spin transition was calculated from the difference spectra using the extinction coeffi cient of ⌬ 390-420 = 110 mM …”

“…The highest intrakingdom diversity in the substrate preferences has been observed in CYP51s from Trypanosomatidae (CYP51E), thus, they share >70% amino acid identity. Thus, the enzyme from Trypanosoma (T) brucei is strictly specifi c toward C4-monomethylated sterols ( 21 ), CYP51 from Leishmania (L) infantum prefers C4-monomethylated sterols but can metabolize lanosterol ( 22 ), and T. cruzi CYP51 prefers C4-dimethylated eburicol ( 23 ). The differences are largely connected with a single amino acid substitution in the B ′ helix [also known as cytochrome P450 substrate recognition site (SRS) 1 ( 24 )], the plant-specifi c F105 in T. brucei and L. infantum versus I105 in T. cruzi (animal and fungal CYP51s all having L in this position) ( 25 ).…”

“…Recent determination of eukaryotic CYP51 structures ( 22,(32)(33)(34) has shown that, contrary to substrate promiscuous P450s, CYP51s do not display any signifi cant structural rearrangements, either upon azole binding or across species and even kingdoms ( 35 ), all having strictly the same set, length, and spatial location of the secondary structural elements [24 helices and 12 ␤ strands separated by 30 loops ( 33 )]. This led us to the suggestion that structural rigidity provides molecular basis for CYP51 functional the substrate and releases one water molecule ( 11,12 ).…”

New Promising Compounds with in Vitro Nanomolar Activity against Trypanosoma cruzi

“…The high selectivity of compounds 1, 3, and 5 toward T. cruzi over T. brucei and L. donovani is probably due to the active site volume and surface area smaller in T. brucei and L. donovani than in T. cruzi that could favor their access in the active site. 20 The aminopyridines 6−8 do not show an interesting CYP51 Tc inhibitory activity, probably because of the lack of the hemeiron coordination (see Figure S4 in Supporting Information). A preliminary study on hepatic metabolism has been carried out on the most active (S)-1 and (S)-3.…”