Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family

Sanglard, Dominique; Loper, John C.

doi:10.1016/0378-1119(89)90014-0

Cited by 91 publications

(51 citation statements)

References 45 publications

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“…The alignment showed a lower identity in the N-terminal region than in the distal extreme. The three conserved regions helix-I, the site for alkane-binding (Sanglard & Loper, 1989;Yadav & Loper, 1999), helix-K and HR-2 (haem-binding) are boxed in Supplementary Fig. S2.…”

Section: Deduced Proteinsmentioning

confidence: 99%

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana

et al. 2010

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The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C 33 as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases. INTRODUCTIONUnlike other insect-pathogenic micro-organisms which must be ingested to initiate disease (virus, bacteria, nematodes and protozoa), entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, mostly invade target hosts by penetrating through their cuticle.The outermost insect surface or epicuticle is covered by a lipid-rich layer, usually composed of very long-chain hydrocarbons together with variable amounts of fatty alcohols, fatty acids and wax esters. The epicuticle protects the insect from desiccation, chemical and biological attack, and acts as a platform for host semiochemicals (Blomquist et al., 1987;Figueiras et al., 2009;Juarez, 1994). Entomopathogenic fungi have the ability to degrade insect cuticular lipids with hydrocarbons, the preferred components capable of supporting fungal growth (Napolitano & Juarez, 1997). The first evidence of the complete catabolism of insect-like hydrocarbons by entomopathogenic fungi was obtained from B. bassiana and M. anisopliae (Crespo et al., 2000). In addition, alkane-grown B. bassiana showed enhanced virulence, by both increasing mortality, i.e. a greater proportion of target hosts killed over time (Crespo et al., 2002), and reducing the time to kill its insect hosts (Pedrini et al., 2009). Although several aspects of the biochemistry of the interaction between entomopathogenic Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, AhR ...

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Section: Deduced Proteinsmentioning

confidence: 99%

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana

et al. 2010

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“…Corresponding P450 genes or cDNAs have been isolated from the alkane-assimilating yeasts Candida maltosa (1)(2)(3)(4)(5)(6), C. tropicalis (7)(8)(9), and C. apicola (10). The presently available 21 sequences belong to the family CYP52, according to the nomenclature of the P450 superfamily (11).…”

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confidence: 99%

“…C]hexadecanoic acid with specific activities of 57, 54, and 56.6 mCi/mmol, respectively, were purchased from either Sigma (Deisenhofen, Germany) or Amersham-Buchler (Braunschweig, Germany). [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]Hexadecanal was produced by enzymatic oxidation of labeled 1-hexadecanol using the peroxisomal fatty alcohol oxidase from the yeast C. maltosa as described previously (14 C]hydroxyhexadecanoic acid were prepared by the P450 52A3-catalyzed hydroxylation of labeled 1-hexadecanol in a reconstituted system composed as described below.…”

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confidence: 99%

Oxygenation Cascade in Conversion of n-Alkanes to α,ω-Dioic Acids Catalyzed by Cytochrome P450 52A3

Scheller¹,

Zimmer²,

Becher³

et al. 1998

Journal of Biological Chemistry

155

119

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Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1,16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1,16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high V max values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min ؊1 , respectively), whereas the oxidation of hexadecanoic acid, 1,16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min ؊1 , respectively). 1-Hexadecanol was found to be the main branch point between mono-and diterminal oxidation. Taken together with data on the incorporation of 18 O 2 -derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono-and diterminal monooxygenation reactions from n-alkanes to ␣,-dioic acids with high regioselectivity.The capability of several yeast species to use n-alkanes and other aliphatic hydrocarbons as a sole source of carbon and energy is mediated by the existence of multiple microsomal cytochrome P450 forms. Corresponding P450 genes or cDNAs have been isolated from the alkane-assimilating yeasts Candida maltosa (1-6), C. tropicalis (7-9), and C. apicola (10). The presently available 21 sequences belong to the family CYP52, according to the nomenclature of the P450 superfamily (11).The metabolic functions of P450 in these yeasts have been established thus far in the terminal oxidation of long-chain n-alkanes to fatty alcohols as the first and rate-determining step of the n-alkane degradation pathway and in the -hydroxylation of fatty acids, initiating the diterminal degradation pathway (Refs. 12-14; for a review, see Ref. 15). Sequential gene disruption revealed that in C. maltosa, four of its eight CYP52 genes, namely CYP52A3, CYP52A4, CYP52A5, and CYP52A9, are directly involved in alkane assimilation (16). After heterologous expression in Saccharomyces cerevisiae, each of the corresponding P450 isoenzymes was found to exhibit an individual substrate specificity in terms of the preferred class (n-alkanes or fatty acids) and the chain length of the hydroxylated aliphatic compounds (17,18).In the present study, we addressed the question of whether P450 monooxygenases may be involved not only in the primary hydroxylation reactions mentioned above but also in the subsequent oxidation steps leading to the formation of fatty acids and long-chain ...

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“…The last item might also indicate that the genomic DNA sequence of P450alk of C. rnaltosa does not contain any intron,since it was suggested previously that the genomic sequence of P-450alk of C. tropicalis does not have any intron. 12 ) This suggestion is supported by the fact that in the ORF there is noTACTAAC sequence which is found typically in the middleofintrons of the genes from S. cerevisiae. 53 ) Most of the members of the P-450 superfamily so far analyzed are membrane-associated proteins in the ER, and so is the case of P-450alk.…”

Section: Alkmentioning

confidence: 50%

Purification of Cytochrome P-450«/A from n-Alkane-grown Cells ofCandida maltosa, and Cloning and Nucleotide Sequencing of the Encoding Gene

Takagi¹,

Ohkuma²,

Kobayashi³

et al. 1989

Agricultural and Biological Chemistry

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Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family

Cited by 91 publications

References 45 publications

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana

Oxygenation Cascade in Conversion of n-Alkanes to α,ω-Dioic Acids Catalyzed by Cytochrome P450 52A3

Purification of Cytochrome P-450«/A from n-Alkane-grown Cells ofCandida maltosa, and Cloning and Nucleotide Sequencing of the Encoding Gene

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