Presencia comercial - (Publicado en el DOF el 15 de diciembre de 2008)

Alignment of CYP6AB11 with the available mammalian P450 templates indicated that the template with the highest sequence identity for CYP6AB11 was CYP3A4 (31% sequence identity). The CYP3A4 template was, in fact, used to build the original D. pastinacella CYP6AB3 model (Mao et al., 2006b) and we used it as the template to predict the CYP6AB11 structure. At the level of primary sequence alignments, CYP6AB11 is 55 % identical to CYP6AB3 and the molecular models of both proteins are structurally similar. Overlays of their active sites (Fig. 5) indicate that most of the active site residues are conserved, particularly those close to the heme, where they form a doughnut-like structure similar to that described in the CYP6AB3 molecular model (Mao et al. 2006b). Even so, CYP6AB11 and CYP6AB3 do not exactly align since there are amino acid insertions of Pro105 (in SRS1) and Pro235 (between SRS2 and SRS3) in CYP6AB3. Interestingly, all of the amino acids creating the doughnut-like structure constricting the catalytic site are conserved except for Ile310 (CYP6AB3) vs. Val308 (CYP6AB11) (Fig. 5). Docking of

as in CYP6AB3 but with a slightly higher interaction energy of 38.4 kcal/mol and at a slightly greater distance from the heme estimated to be 7.2 Å. The Ile310-to-Val308 replacement in CYP6AB11 enlarges the opening to the heme and is predicted to allow imeratorin to be slightly more mobile than in the CYP6AB3 catalytic site.

4. Discussion

Without a fully sequenced genome, characterizing the xenobiotic-

metabolizing P450 inventory of a particular species is experimentally challenging. Our effort to identify the subset of P450s in the NOW genome associated with xenobiotic metabolism, however, was greatly facilitated by the use of bioinformatics, homology modeling, and comparative ecology, which allowed us to build on prior work identifying substrate specificities in related P450s. Thus, we were able to demonstrate that CYP6AB11 is able to metabolize imperatorin at a significant rate (0.88 pmol/min/pmol P450). Imperatorin is a toxic furanocoumarin that has been isolated from a range of plant species in the family Apiaceae (Berenbaum, 1991; Berenbaum and Zangerl, 1998; Baek et al., 2000). While NOW has no known apiaceous hostplants, it is commonly found on fig (Ficus carica), a moraceous plant reported to contain several linear furanocoumarins, including both oxypeucedanin and oxypeucedanin hydrate, which, like imperatorin, contain an 8-O-prenylated side chain (Zaynoun et al., 1984; Towers, 1986). In other species, furanocoumarins with an extended side chain at the 8 position are metabolized by different P450s from those which metabolize 8- or 5-methoxy-substituted furanocoumarins. For example, whereas CYP6B1 in P. polyxenes, the black swallowtail, metabolizes both

xanthotoxin (8-methoxypsoralen) and bergapten (5-methoxypsoralen) at high rates, it apparently has little to no catalytic activity against imperatorin (Cohen et al. 1992). Conversely, CYP6AB3 from D. pastinacella, which is highly active against

imperatorin, has little to no catalytic activity against either bergapten or xanthotoxin (Mao et al., 2006b, 2007b). Among lepidopteran furanocoumarin-metabolizing P450s, only CYP6B4 from the polyphagous P. glaucus can metabolizes imperatorin as well as 5-methoxy substituted furanocoumarins, possibly because several amino acid

a greater range of substrates (Li et al., 2003).

CYP6AB11 shares 55% identity with CYP6AB3 and, based on a comparison of their predicted 3-dimensional structures, also shares a doughnut-like structure immediately above the heme that limits substrate access to the catalytic site. Key residues limiting access include Phe118 in SRS1 and Ala314 and Thr318 in SRS4 and Leu380 in SRS5, which are conserved both in CYP6AB3 and CYP6AB11 (Fig. 5) (Mao et al., 2006b). This doughnut structure in CYP6AB11 and CYP6AB3 has not been found in other CYP6 family proteins sequenced to date. The imperatorin metabolite generated by CYP6AB11 possesses an epoxide side chain, the result of attachment of oxygen to the side chain double bond; the CYP6AB11 model docked with imperatorin shows that this substrate can bind in the same orientation as it does in CYP6AB3. Comparison of the predicted interaction energies shows that in imperatorin would bind in the CYP6AB11 catalytic site with a slightly higher interaction energy consistent with the metabolic assay results. The protein alignment within the CYP6AB families reveals that CYP6AB11 does not have an Ala92Val replacement that characterizes the CYP6AB3v2 variant, which is predicted to greatly increase imperatorin metabolism by increasing electron transfer between P450 and P450 reductase (Mao et al., 2007b). The absence of this replacement helps to explain why the catalytic activity of CYP6AB11 is closer to that of CYP6AB3v1 than that of CYP6AB3v2.

PBO is known as a general P450 inhibitor and has historically been used as a synergist applied with pyrethroid insecticides to control insects. It has two rings plus a long side chain (C9H19O3) as well as a short side chain (C3H7). Mechanisms of PBO metabolism are complex and not fully clear; most studies were completed with mammalian P450s have identified more than 10 metabolites, but the specific

biochemical pathways leading to their formation have not yet been fully elucidated (Byard and Needham, 2006). Although CYP6AB11 exhibits low levels of activity toward PBO, we were unable to detect a metabolite by GC-MS. Surprisingly, CYP6AB11 displayed no consistent detectable activity against myristicin, another

myristicin levels declined less than 5% in the metabolism reactions incubated for 2 hr. In contrast, CYP6AB3v2 actively metabolizes myristicin (Mao et al., 2008b).

Because the disappearance rate is close to the detection limit and because the disappearance rate is highly variable across replicates, it is still not clear whether myristicin can be metabolized by CYP6AB11.

Although CYP321A1 from H. zea displays activity against a very broad range of phytochemical, insecticide and mycotoxin substrates (Sasabe et al., 2004; Rupasinghe et al., 2007; Niu et al., 2008), using similar techniques I was unable to demonstrate metabolic activity of CYP321C1 against any of the 16 tested chemicals. The protein alignment of CYP321C1 with CYP321A1 shows that there is a high level of amino acid identity at the back of the catalytic site in the I helix (SRS4) and the loop sequences above the catalytic site (SRS3), but there are also many divergent amino acid residues in other SRS regions which might be critical for substrate channel architecture. Our docking models show that CYP321A1 has a spacious cavity allowing larger and more rigid molecules, such as aflatoxin B1, to access its catalytic sites.Compared with the CYP321A1 molecular model, CYP321C1 has a smaller catalytic core structure which may block access to the catalytic sites for larger molecules (data not shown).

Only two compounds, xanthotoxin and aflatoxin B1, were examined as possible substrates for CYP6B44, while previous work with P. polyxenes CYP6B1 suggested some degree of specialization for furanocoumarin metabolism. No metabolism of either compound could be detected with CYP6B44. Amino acids critical for metabolism of furanocoumarins in the SRS domains of CYP6B proteins include Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6, which are predicted to form a network that stabilizes the catalytic sites and allows the furanocoumarins to reach its catalytic core (Chen et al., 2002; Baudry et al., 2003). Among them, Phe116 in SRS1 is conserved in all CYP6B proteins and His117 is conserved except in CYP6B44 where Asn117 replaces His117. The other amino acids including Phe371 in SRS5 and Phe484 in SRS6 are divergent between different

Substitution of Ile115 for Leu115 in SRS1 of CYP6B1 can greatly enhance its activity via changes in the metabolite release channel (Pan et al., 2004; Wen et al., 2005). The fact that the Ile115Leu replacement is found in CYP6B44 should facilitate furanocoumarin metabolism. CYP6B8 can metabolize more diverse chemicals than the specialized furanocoumarin metabolizing P450s. The molecular model built by Li et al. (2004b) shows that CYP6B8 has a larger and more flexible catalytic site than CYP6B1 and also has one more substrate access channel than CYP6B1.

The presence of a seemingly fairly specialized CYP6B11 in the broadly polyphagous NOW appears to contrast with previous findings that CYP6B genes in polyphagous lepidopterans are as a rule broadly substrate-specific. Our effort to identify more substrates by subjecting a whole-plant extract to metabolism assays (cf., Mao et al., 2008b) failed to reveal any additional suitable substrates, despite

considerable chemical complexity present in these extracts. This seemingly specialized enzyme may be a historical vestige of a closer association with a particular subset of hostplants. Navel orangeworms, as the name suggests, were originally described from Citrus spp. (Mote, 1922), although such host use is rarely reported today (Mahoney et al., 1989). It is intriguing to note, however, that citrus and other rutaceous species are rich in furanocoumarins and may have at some point in the evolution of this species represented a major host and selective agent on metabolic detoxification pathways. An examination of the host use patterns and detoxification capacity of the five congeners of A. transitella may shed light on the significance of furanocoumarins in the evolution of this pest species.

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Table 1 Primers used for 5’ and 3’ RACE amplification of P450s from Amyelois

transitella

Primers Sequences (5’ to 3’)

FDPER primer GCGGATCCTT(T/C)GA(T/C)CC(I/A)GA(A/G)AG(A/G)TT C3PT GAC TCG AGT CGG ATC CAT CGT TTT TTT TTT TTT TTT T C3 GAC TCG AGT CGG ATC CAT CG

62SM-GSP1 CAG CAC AGC CGC CAA TCC AGC CAA T 62SM-NGSP1 TAGGTCCCGTACCAAAGGGCAGATAAAT 34SM-GSP1 GGGCGAATCTCATACCGACGCATAGTCT 34SM-NGSP1 TACCGACGCATAGTCTGTTTCCTTTACC 55SM-GSP1 GCACACTCGGCTCTGGACTTTGGCG 55SM-NGSP1 TGGTCCGACCCCAAAAGGTATATAAGCG GN6AB11_CDS_forward GGCGGTCCGGAATTCATGATCATACCAATTGCCGTAATTG Rsr II Ecor I GN6AB11_CDS_reverse GGGCGGCCGCAAGCTTTTAATTAAAATTGACCACATTTCGC Not I Hind III

GN66B44-CDS_forward GGTCTAGACTGCAGATGATTCCCTTTATTTTGGTGCTA ATAAC Xba I Pst I

GN6B44_CDS_reverse GGCTCGAGAAGCTTTTAATACCTAGGGTTGAACTTCAAATGC XhoI Hind III

GN321C1-CDS_forward GGGTCTAGAATGTTTGTCCAGATAATACTGTTAATTTTTATTG Xba I

GN321C1_CDS_reverse GGCTCGAGAAGCTTTTATATGTTTCTCCAAATGAATTCAATATC Xho I Hind III

Table 2 Analytical methods for the substrates of the metabolism assays. Substrates Internal

standard

Analysis method

Elution solvent Detec

tion (nm) Eluti on time (min) xanthotoxin bergapten Normal

HPLC