Molecular cloning and characterization of the primary structure of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa

Schunck, Wolf‐Hagen; Kärgel, Eva; Gross, B; Wiedmann, Brigitte; Mauersberger, Stephan; Kopke, Katarina; Kießling, U.; Strauß, Michael; Gaestel, Matthias; Müller, Hansgeorg

doi:10.1016/0006-291x(89)92677-6

Cited by 50 publications

(18 citation statements)

References 25 publications

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“…ox. [13]), CYP3All from mouse (mouse [14]), CYP3A4 from human (human [15]), and CYP56 from the yeast Candida maltosa (yeast [16]). Sequences were aligned using the BLASTX program from the National Center for Biotechnology Information.…”

Section: ~--Ff]lic L U a P ' A R L • A~i ^Ddementioning

confidence: 99%

Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus

et al. 1996

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Section: ~--Ff]lic L U a P ' A R L • A~i ^Ddementioning

confidence: 99%

Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus

et al. 1996

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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%

“…For that purpose, P450 52A3, 1 which represents the major alkane-inducible P450 form in C. maltosa (16,19), was selected. Its substrate specificity is characterized by a preferential binding and hydroxylation of n-alkanes compared with fatty acids and by a general chain length optimum for such compounds with 16 carbon atoms (3,17).…”

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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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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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“…Based on previous studies in C. tropicalis (17,21) and C. maltosa (19), it was predicted that the CYP52 family would contain numerous members. This was confirmed in this study by the isolation of 10 CYP52 genes.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of the CYP52 Family of Candida tropicalis ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids

Craft

Madduri

Mark

et al. 2003

Appl Environ Microbiol

133

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Candida tropicalis ATCC 20336 excretes ␣,-dicarboxylic acids as a by-product when cultured on n-alkanes or fatty acids as the carbon source. Previously, a ␤-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the -hydroxylase enzymes catalyzing the first step in the -oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of -oxidation of n-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (CYP52) of C. tropicalis ATCC20336 as well as the accompanying NCP were cloned and sequenced. The 10 CYP genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes, CYP52A13 and CYP52A14 showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair CYP52A17 and CYP52A18 was also induced under all three conditions but to a lesser extent. Moderate induction of CYP52A12 was observed. These results identify the CYP52 and NCP genes as being involved in ␣,-dicarboxylic acid production by C. tropicalis and provide the foundation for biocatalyst improvement.␣,-Dicarboxylic acids (␣,-diacids) are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives, and macrolide antibiotics. Currently, only three diacids, nonanedioic (azelaic), decanedioic (sebacic), and dodecanedioic, are available at a quantity and cost acceptable for commercial applications. Long-chain diacids with more than 12 carbons offer potential advantages over shorter-chain diacids, but their limited commercial availability and high price have prevented widespread growth in many of these applications.Several species of yeasts belonging to the genus Candida excrete ␣,-diacids as a by-product when cultured on n-alkanes or fatty acids as the carbon source (22). One such yeast, Candida tropicalis ATCC 20336, is the subject of this paper.In Candida spp., n-alkanes and fatty acids are metabolized by enzymes present in the ␤-oxidation and -oxidation pathways. For alkanes, the first reaction occurs in the -oxidation pathway with the formation of the corresponding alcohol. This reaction is catalyzed by a cytochrome P450 hydroxylase complex, which consists of a cytochrome P450 monooxygenase (the CYP protein) and the accompanying NADPH cytochrome P450 reductase (NCP). A terminal carboxy function is ultimately formed from two additional oxidation steps, catalyzed by a fatty alcohol oxidase and a fatty aldehyde dehydrogenase. Fatty acid fo...

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Molecular cloning and characterization of the primary structure of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa

Cited by 50 publications

References 25 publications

Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus

Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus

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

Identification and Characterization of the CYP52 Family of Candida tropicalis ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids

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