Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol

Sandstrom, Pamela; Welch, William H.; Blomquist, Gary J.; Tittiger, Claus

doi:10.1016/j.ibmb.2006.08.004

Cited by 97 publications

(97 citation statements)

References 42 publications

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“…Cells were lysed and microsomes prepared as per ref. 44. Production of P450-CPR fusion protein was determined by Western blotting using a polyclonal anti-CPR antibody (40) diluted 1:1,000 with a 1:5,000 dilution of HRP-conjugated goat anti-rabbit secondary antibody (BioRad) and SuperSignal West Pico (Pierce) chemiluminescent substrate (Fig.…”

Section: Methodsmentioning

confidence: 99%

An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis

Qiu

Tittiger

Wicker-Thomas

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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371

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Insects use hydrocarbons as cuticular waterproofing agents and as contact pheromones. Although their biosynthesis from fatty acyl precursors is well established, the last step of hydrocarbon biosynthesis from long-chain fatty aldehydes has remained mysterious. We show here that insects use a P450 enzyme of the CYP4G family to oxidatively produce hydrocarbons from aldehydes. Oenocyte-directed RNAi knock-down of Drosophila CYP4G1 or NADPH-cytochrome P450 reductase results in flies deficient in cuticular hydrocarbons, highly susceptible to desiccation, and with reduced viability upon adult emergence. The heterologously expressed enzyme converts C 18 -trideuterated octadecanal to C 17 -trideuterated heptadecane, showing that the insect enzyme is an oxidative decarbonylase that catalyzes the cleavage of long-chain aldehydes to hydrocarbons with the release of carbon dioxide. This process is unlike cyanobacteria that use a nonheme diiron decarbonylase to make alkanes from aldehydes with the release of formate. The unique and highly conserved insect CYP4G enzymes are a key evolutionary innovation that allowed their colonization of land. Insects are the largest group of extant terrestrial organisms. As their crustacean ancestors moved from aquatic to terrestrial environments, insects were confronted with a new, dry frontier. Insects solved the ecophysiological problem of how to restrict water loss to prevent dessication by depositing long-chain hydrocarbons as essential waterproofing components on their epicuticle (1). These diverse chemicals now serve many additional functions, particularly in defense, reproduction, and communication (2). In flies, cuticular hydrocarbons (CHs) are a complex blend of long-chain (∼C21-C37+) alkanes and alkenes that serve as species-and sex-specific semiochemicals, and some components are sex pheromones. CHs also serve in nest mate recognition by social insects and as trail pheromones in ants; the complexity of their blend can be useful in taxonomic discrimination of mosquitoes. Much is known about the biosynthesis of CHs from fatty acids in insects, involving a complex network of fatty acid synthases, elongases, and desaturases, leading to very long-chain acyl-CoA thioesters. These are converted by acyl-CoA reductases to aldehydes that serve as substrates for the last oxidative decarbonylation step (2) (Fig. 1). The single carbon chain-shortening conversion of precursor aldehydes to hydrocarbons is catalyzed by a P450 enzyme, P450hyd, with release of CO 2 (3), but this enzyme has not yet been identified. The only known decarbonylase that has recently been described occurs in cyanobacteria that use a nonheme diiron enzyme (4-8) to catalyze the last step (Fig. 1). Insect CH are synthesized in large ectodermally derived cells (9) called oenocytes (10-12), and are then shuttled by hemolymph lipophorin (13,14) to the cuticle, where they are deposited on the outer epicuticular layer. Here we identify P450hyd as CYP4G1 in Drosophila melanogaster, and show that the enzyme is massively coexpresse...

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Section: Methodsmentioning

confidence: 99%

An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis

Qiu

Tittiger

Wicker-Thomas

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

371

405

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“…Cytosolic actin mRNA, previously used in the Ips genus as a stable endogenous control for transcription experiments (Keeling et al, 2006;Sandstrom et al, 2006), was used as a normalizer to calculate the relative mRNA abundance. As shown in Figure 7, AmyA was expressed at a significantly higher level than AmyB1 in imagoes (Po0.01), larvae (Po0.05) and adult abdomens (Po0.05).…”

Section: The Mrna Expression Of A-amylase Paralogsmentioning

confidence: 99%

Characterization of two closely related α‐amylase paralogs in the bark beetle, Ips typographus (L.)

Viktorinová

Kučerová

Bohmova

et al. 2011

Arch Insect Biochem Physiol

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Ips typographus (L.), the eight-spined spruce bark beetle, causes severe damage throughout Eurasian spruce forests and suitable nuclear markers are needed in order to study its population structure on a genetic level. Two closely related genes encoding a-amylase in I. typographus were characterized and named AmyA and AmyB. Both a-amylase paralogs consisted of six exons and five introns. AmyA encodes a polypeptide of 483 amino acids, whereas AmyB has two alternative transcripts encoding polypeptides of 483 and 370 amino acids. The expression levels of both genes were high during larval stage and adulthood. The AmyB transcripts were absent in the pupal stage. A modification of the allozyme staining method allowed us to detect two clusters of bands on the electrophoretic gel that may correspond to the two a-amylase genes. There was a correlation between the lack of AmyB expression in pupa and the absence of the fast migrating isozyme cluster at this stage, suggesting that the faster migrating isoforms are products of the AmyB gene, whereas the slowly migrating bands are derived from the AmyA. C 2011 Wiley Periodicals, Inc.

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“…Microbial biocatalysts often specifically hydroxylate inactive sites, such as a methylene moiety, 23,24 which facilitates synthesis of more derivatives from the resultant hydroxyl moiety. With its aromatic ring hydroxylated by A. azurea JCM-3275 to add the active moiety, AS1429716 is a novel immunosuppressive drug candidate.…”

Section: Discussionmentioning

confidence: 99%

Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392

et al. 2010

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Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209 bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.

show abstract

Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol

Cited by 97 publications

References 42 publications

An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis

An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis

Characterization of two closely related α‐amylase paralogs in the bark beetle, Ips typographus (L.)

Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392

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