The use of biotin-streptavidin systems for enhancing the parameters of immunometric analysis

The possibility of improving analytical parameters of the immunometric assay with the use of biotinylated antibodies and biotin-streptavidin complexes in comparison with the commonly known approach of direct antibody modification with 125I has been studied. Experiments have been carried out with the use of low-affinity antibodies (Kass approximately 10(9) M-1) to ferritin. The signal-to-noise ratio in the immunometric increases 2.3 times when streptavidin labeled with horse-radish peroxidase is used and 4.3 times when the preformed streptavidin + biotin-peroxidase complex is used in comparison with assay systems based on 125I-labeled antibodies. The improvement of assay parameters of immunochemical systems by means of biotin-streptavidin complexes has been found to permit the use of low-affinity antibodies as assay reagents, thus ensuring analytical parameters attaining or close to those of immunoradiometric assay systems based on high-affinity 125I-labeled antibodies (Kass approximately 10(10) M-1). As shown in this study, the following factors ensure the signal enhancement in biotin-streptoavidin systems: (a) the biotin modification of several lysin residues per IgG molecule, the optimum extent of modification being 3-4 residues per molecule; (b) mild procedure for biotinylation. In contrast to oxidative iodination, the modification of NH2 groups with biotin esters does not significantly affect their antigen-binding properties.     

Low Levels of Genetic Diversity within Populations of Hosta clausa (Liliaceae)

Abstract Genetic diversity of Korean populations in Hosta clausa was investigated using starch gel electrophoresis. Hosta clausa is widespread, grows only along streamsides, and has both sexual and asexual reproduction. Populations of the species are small and isolated. Thirty-two percent of the loci examined were polymorphic, and mean genetic diversity within populations (He_p=0.082) was lower than mean estimates for species with very similar life history characteristics (0.131), particularly for its congener H. yingeri (0.250). The mean number of multilocus genotypes per population was 8.7, and genotypic diversity index (D_G) was 0.84. Significant differences in allele frequencies among populations were found in all seven polymorphic loci (P < 0.001). About one-fifth of the total allozyme variation was among populations (G_ST=0.192). Indirect estimate of the number of migrants per generation (Nm=0.48, calculated from mean G_ST) and nine private alleles found indicate that gene movement among populations was low. The low levels of genetic diversity within populations and the relatively high levels of genetic diversity among populations suggest that strong moist habitat preferences, clonal reproduction, low level of gene flow among populations, genetic drift, and historical events may have played roles in the genetic structuring of the species.     

Cassava leaf harvesting as vegetables; a cause of vulnerability of cassava plant to cassava mosaic disease and eventual yield reduction: Ernte von cassavablättern als gemüse; eine ursache für die anfälligkeit der cassavapflanze für die cassava-mosaikkrankheit und für spätere ertragseinbußen

The consequence of harvesting young leaves of cassava as vegetable on the vulnerability of the crop to cassava mosaic disease (CMD) and on storage root yield was investigated using 30 cassava genotypes planted in IITA fields located in the humid forest (Port Harcourt?:?Onne), forest-savannah transition (Ibadan), southern guinea savannah (Mokwa) and northern guinea savannah (Zaria) agroecologies in Nigeria. Tender apical leaves and shoots of the cassava genotypes were removed from forty plants per cassava genotype with the same number of plants considered as control. Whitefly infestation, disease incidence (DI) and symptom severity (ISS) of the disease were assessed at monthly interval for six months and also at the ninth month after planting (MAP). Yield reduction due to this treatment was calculated as percentage harvest index (HI). Whitefly population fluctuated throughout the period of observation at all locations with higher population obtained generally for treated plants compared to control plants. Sprouting leaves of some treated genotypes were observed with severe mosaic symptoms, while corresponding control showed no mosaic symptoms. Contrarily, no remarkable difference was observed in Zaria between the mean ISS of treated and control cassava genotypes. There was a highly significant difference (P?<?0.01) in DI and ISS among cassava genotypes across all locations. Also, there was a highly significant interaction (P?<?0.01) in symptom severity between location (loc) and genotype, genotype and treatment (trt), loc and trt. Interaction between loc, genotypes and trt with regard to DI was highly significant at 2, 3 and 4 MAP, while with ISS, the interaction was highly significant all through the counting period. There was a positive relationship between DI and ISS on plants of genotypes 96/1039 and ISU. The percentage HI (27.4) of treated plants of genotype 95/0166 in Ibadan was remarkably lower than the value obtained for corresponding control (41.9) plants. Also, sharp distinction in% HI of treated (39.5) and control (43.8) ISU was observed in Onne with their respective ISS values as 3.7 and 3.2. Therefore, harvesting tender apical leaves and shoots of cassava as vegetables should be discouraged as it increases the severity of CMD infection in the regenerating shoots of cassava with attendant storage root yield reduction.     

Seasonal mortality trends in tree‐feeding insects: a field experiment

Abstract 1. The majority of general life‐history models treat the environment as being invariable through time, even though temporal variation in selective agents could dramatically change the outcomes, e.g. in terms of optimal size and time at maturity. For herbivorous insects, seasonal differences in food quality are reasonably well described, but seasonal dynamics of top‐down selective forces are poorly documented. 2. The present study attempted to quantify seasonal changes in predation risk of folivorous insect larvae in temperate forest habitats. In a series of field experiments, artificial larvae were exposed to predators, and the resulting bird‐inflicted damage was recorded. The trials were repeated regularly throughout the course of two summers. 3. A distinct peak of larval mortality was recorded in mid‐June (the nestling period for most insectivorous passerine birds), after which predation risk declined to a plateau of 20–30% below the peak value. 4. The recorded pattern is interpreted as a consequence of seasonal changes in the number and behaviour of insectivorous birds, and the abundance of alternative food resources for these predators. 5. A quantitative analysis based on field data indicated that considering temporal variation in mortality in life‐history models is crucial for obtaining realistic predictions concerning central life‐history traits, such as final body size in different generations.     

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.     

Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

14alpha-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC(50) for fluconazole, suggesting that F145 (conserved only in fungal 14alpha-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.     

Crystal Structures of Trypanosoma brucei Sterol 14��-Demethylase and Implications for Selective Treatment of Human Infections

Sterol 14α-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.     

A second FMN binding site in yeast NADPH-cytochrome P450 reductase suggests a mechanism of electron transfer by diflavin reductases

NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between two semiquinone forms, neutral (blue) and anionic (red).     

Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum

Leishmaniasis is a major health problem that affects populations of ～90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14α-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14α-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 ～10 and 8 min(-1), respectively), it is also found to 14α-demethylate C4-dimethylated lanosterol (V(max) = 0.9 min(-1)) and C4-desmethylated 14α-methylzymosterol (V(max) = 1.9 min(-1)). Binding parameters with six sterols were tested, with K(d) values ranging from 0.25 to 1.4 μM. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14α-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. 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.     

The First Virally Encoded Cytochrome P450

The genome sequence of the giant virus Acanthamoeba polyphaga mimivirus revealed the presence of two putative cytochrome P450 (CYP) genes. The product of one of the two predicted CYP genes ({"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162) showed low-level homology to sterol 14-demethylase (CYP51) and contained a C-terminal polypeptide domain of unknown function. {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 expression (without an N-terminal membrane binding domain) in Escherichia coli yields a CYP protein which gives a reduced CO difference maximum at 448 nm and was formally demonstrated as the first viral cytochrome P450. Analysis of binding of lipid and sterol substrates indicated no perturbation in CYP heme environment, and an absence of activity was seen when 14-methyl sterols were used as a substrate. The function of the CYP protein and its C-terminal domain remain unknown.Cytochromes P450 (CYP) are a superfamily of heme-thiolate enzymes that are distributed widely throughout Eukarya, Archaea, and Bacteria (http://drnelson.utmem.edu/CytochromeP450.html). Viruses are the most abundant biological entities in nature and are also responsible for many diseases in plants and animals. To date, 2,180 viral genome sequencing projects have been completed and annotated and no CYP open reading frames have been observed (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=10239&opt=Virus).Acanthamoeba polyphaga mimivirus is the largest known virus, which grows in amoeba (5). In 2004, the 1.2-Mbp genome of mimivirus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY653733","term_id":"55416625","term_text":"AY653733"}}AY653733) was sequenced (9). Its genome is larger than that of several bacteria and archaea and is predicted to encode 911 proteins, among which only 298 have predicted functions. Many atypical proteins are predicted to be encoded by the mimivirus genome, including key protein translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (9). Interestingly, among genes never yet reported to occur in a virus, mimivirus contained two putative gene sequences predicted to encode cytochrome P450 enzymes (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 and {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162, also known as MIMI_L532 and MIMI_L808, respectively). First, {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 is a putative protein of 468 amino acids in length. In a BLASTP search, this putative CYP protein showed homology to a range of bacterial P450 proteins, including a P450 protein from Chloroflexus aurantiacus (23% identity) and CYP171 from Streptomyces peucetius (23% identity). Additionally, {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 also showed homology at the same level to nematode P450 proteins, including Caenorhabditis briggsae CYP37B1 (25% identity) and a P450 protein from the sea squirt Ciona intestinalis similar to the CYP4 family (24% identity). Efforts in our laboratory to express the {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 gene and verify that it indeed encodes a cytochrome P450 have been unsuccessful, but additional attempts are in progress. The mimivirus protein {"type":"entrez-protein","attrs":{"text":"YP_143886","term_id":"55980589","term_text":"YP_143886"}}YP_143886 was designated CYP5254A1 by David Nelson (http://drnelson.utmem.edu/CytochromeP450.html).The second putative mimivirus CYP protein ({"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162) showed in a BLASTP search the strongest homology to CYP51 proteins (7) from a variety of organisms, including protozoal CYP51 proteins from, e.g., Leishmania major (23% identity); plant CYP51 proteins from, e.g., Arabidopsis thaliana (22% identity); and fungal CYP51 proteins from, e.g., Aspergillus fumigatus (21% identity). This homology is low, strongly suggesting the absence of a functional link. Further analysis of the {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 709-amino-acid sequence revealed this putative CYP protein to be approximately 200 residues longer than its closest CYP homologues, and this protein was proposed to comprise a fused protein domain of unknown function, with the best homologies to lipopolysaccharide core biosynthesis glycosyl transferase from Proteus mirabilis HI4320 (26% identity), integral membrane sensor signal transduction histidine kinase from Dinoroseobacter shibae DFL 12 (24% identity), a short region of dysferlin from Strongylocentrotus purpuratus (35% identity), and pierisin-1 (NAD-DNA ADP-ribosyltransferase) from Pieris rapae (24% identity). Interestingly, several putative posttranslational modifications, including one N glycosylation site, a protein kinase C phosphorylation site, four casein kinase II phosphorylation sites, and three myristoylation sites, were predicted to exist in this C-terminal extension peptide, representing the first time these specific modifications were present in a P450 molecule.Historically, a protein can be identified as a cytochrome P450 through the production of the carbon monoxide (CO)-bound form of the reduced (sodium dithionite-treated) pigment, which has an intense absorption band at 450 nm (8). Following isopropyl β-d-thiogalactopyranoside-induced expression of the full-length {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 gene, utilizing the T7 promoter of the Escherichia coli expression vector pET17b (Novagen), only a protein producing a Soret maximum at 420 nm, recognized as the misfolded, incorrect form of CYP, was detected in reduced-difference CO spectrophotometry. Alterations in temperature, coexpression with molecular chaperones GroES and GroEL of E. coli (which allow production of active and correctly folded human P450s [4]), and the use of different E. coli strains for recombinant protein expression did not produce correctly folded {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (data not shown). Analysis of the primary sequence revealed the presence of a putative membrane-spanning segment located from residue 2 to 19 which may interfere with the expression of correctly folded P450 (1). A modified {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 gene sequence encoding an insertion of alanine at amino acid position 2 was generated by PCR. This N-terminally truncated enzyme (Fig. ​(Fig.1A),1A), expressed as a correctly folded CYP protein, generated a characteristic reduced-CO-difference spectrum with a maximum at 448 nm (Fig. ​(Fig.1B).1B). Cell fractionation revealed the truncated protein to be associated with the membrane fraction following ultracentrifugation at 100,000 × g, and no CYP protein was detected in the E. coli cytoplasm, thus necessitating the use of detergents to purify the enzyme. The truncated but membrane-bound enzyme was expressed at CYP levels of >1,000 nmol P450/liter of culture, with supplementation of the growth medium with the heme precursor δ-aminolevulinic acid increasing heme-incorporated CYP expression levels approximately twofold, to 2,000 to 3,000 nmol P450/liter of culture. Truncated {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 was the major band observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Fig. ​(Fig.1C),1C), with the molecular mass estimated to be 78 kDa, in agreement with the predicted molecular mass of the truncated protein. The absolute absorbance spectrum of the purified (oxidized, Fe³⁺) {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 protein showed a Soret band at 419 nm and α and β bands at 572 and 536 nm, while reduction with sodium dithionite (Fe²⁺) resulted in a typical Soret peak shift to 417 nm. Most CYP enzymes are purified in a low-spin state with a water molecule hexacoordinated to the CYP heme iron, as indicated by a peak at 390 nm in the absolute spectrum. Substrate or inhibitor addition shifts the heme to the high-spin state, as indicated by a peak at 419 nm in the absolute spectrum, which can be the case when imidazole, used to purify the protein, binds to the heme iron. Continued dialysis for removal of the imidazole from the protein resulted in a shift from a high- to a low-spin state. Furthermore, quantification of the iron content (2) of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (0.97 ± 0.06 atoms of iron per heme-containing molecule of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162) indicated that there is one atom of iron per heme-containing molecule of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162, confirming one atom of iron associated with the heme of this P450 protein. Given the weak homology of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 to sterol-metabolizing CYP proteins, the binding of the sterols lanosterol and obtusifoliol as well as the final A. polyphaga sterol end product ergosterol to purified enzyme was investigated as previously described (3). No evidence of sterol binding or metabolism was obtained (data not shown). Such data can be confirmed by the fact that the key motif aGQHTSs (which is involved in catalysis, includes an invariant H in all CYP51 proteins to date [6], and corresponds to a negatively charged residue [D/E] in other P450 families) is missing in {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (Fig. ​(Fig.2A).2A). Additionally, {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 did not cluster with any CYP51 proteins but mapped to a distinct and separate branch on the tree (Fig. ​(Fig.2B).2B). A homology model was generated for mimivirus {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 protein on the basis of the resolved P450 crystal structures of Mycobacterium tuberculosis CYP51 (Protein Data Bank accession no. 1E9X) and flavocytochrome CYP102A1 from Bacillus megaterium (Protein Data Bank accession no. 1JPZ). {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 is predicted to adopt a typical P450 fold reflecting the similarity in helix assignment. It was possible to confirm the likely heme-coordinating residue (C425), and the EXXR motif, present in nearly all P450 proteins and involved in heme binding and P450 architecture, is also conserved in {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (Fig. ​(Fig.3).3). Consequently, the mimivirus protein {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 was designated CYP5253A1 (http://drnelson.utmem.edu/CytochromeP450.html).Open in a separate windowFIG. 1.Purification and spectral characterization of mimivirus {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162. (A) N-terminal sequence of the full-length native enzyme, with the hydrophobic stretch that most likely forms a transmembrane α helix underlined. Below is shown the N-terminal sequence of the truncated CYP protein used to obtain the correctly folded P450 protein. (B) Absolute oxidized and reduced CO difference (inset) spectra at 1 μM P450 concentration. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (10%). Lane 1, rainbow marker; lane 2, purified mimivirus CYP51-like protein.Open in a separate windowFIG. 2.Alignment of mimivirus {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 with 143 CYP51 family members (only two representatives of each biological kingdom are shown). (A) The fragments shown are the BC loop (SRS1 region, helices F and G [SRS2 and SRS3] and helix I [SRS4]). (B) Representative phylogenetic tree of CYP51 sequences showing the position of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162.Open in a separate windowFIG. 3.Mimivirus P450 model with marked secondary structural elements. The resultant homology models were validated by cross-reference to the secondary structure predictions. Homology models were generated with 10 iterations of the MODELLER program, and the model structure was clipped to the first 480 amino acids, for which the sequence identity with the resolved crystal structures of MTCYP51 and flavocytochrome P450-2 of Bacillus megaterium for the CYP domain was 16%. The energy-minimized model has very good ProsaII and Profiles 3D scores.The presence of genes encoding CYP in the mimivirus genome is intriguing from the standpoint of P450 evolutionary discussion. Phylogenetically, mimivirus and other giant viruses are very old and are thought to have existed prior to cellular organisms (9). Furthermore, it was previously proposed that a form of cytochrome P450 has been present in life forms for billions of years and before the advent of free atmospheric oxygen (10). Subsequently P450 had protective value in detoxifying reactive oxygen species and was retained in aerobic organisms as a monooxygenase in biosynthetic processes and in the degradation of complex molecules. Consequently, it can be speculated that P450 may have been present in a viral genome prior to the establishment of the three domains of life (eukaryotes and prokaryotes, which consist of bacteria and archaea). It is possible that mimivirus acquired CYP genes from a more ancient progenitor. Conversely, Moreira and Brochier-Armanet (7) hypothesize that the diverse mimivirus genes, many with eukaryotic homology, including CYP, were obtained by horizontal gene transfer. The basis of this theory suggests that the mimivirus host, A. polyphaga, is also host to parasitic, bacterial endosymbionts, including many Mycobacteria spp. which have high numbers of CYP genes within their genomes. Consequently, mimivirus may have picked up genes from such endosymbionts or from the amoeba host itself. At present, no Acanthamoeba genome has been sequenced to allow comparisons, but Acanthamoeba genomes certainly contain CYP51 for synthesis of sterols. Finally, although mimivirus was first isolated from A. polyphaga, additional hosts acting as a source of new genetic material for this virus cannot be ruled out. For example, mimivirus has been implicated as a causative agent of influenza in mice and humans, suggesting mammalian cellular hosts for mimivirus. It can also be argued that mimivirus CYP has evolved into a gene encoding a protein with a totally different function, unrelated to its being a P450. It is probable that we will never know the nature of the ancient P450 ancestor or how it evolved into the superfamily that we see today (>8,500 genes). However our confirmation of a viral gene encoding a cytochrome P450 protein invigorates this continuing debate.