The cytochrome P-450 alkane monooxygenase system of the yeast lodderomyces elongisporus: Purification and some properties of the NADPH-cytochrome P-450 reductase

Honeck, Horst; Schunck, Wolf‐Hagen; Riege, P.; Müller, Hansgeorg

doi:10.1016/0006-291x(82)91257-8

Cited by 43 publications

(12 citation statements)

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“…In the proposed n-alkane assimilation pathway, n-alkane is first hydroxylated at the terminal position to produce n-alcohol and then oxidized successively to fatty acids. The growth property of the disrupted strain that could not assimilate n-alkanes but could grow on n-alcohols and fatty acids as sole carbon sources clearly correlated with the in vitro n-alkane-hydroxylating activity of the P450alk enzymes shown here and in previous studies (5,9,12,13), confirming that P450alk catalyzes the first step of n-alkane assimilation. This growth property also indicates that none of the four isoforms analyzed is essential in the further downstream steps of the assimilation pathway, although considerable fatty acid -hydroxylation activity has been shown for some of them (5,(11)(12)(13).…”

Section: Discussionsupporting

confidence: 64%

“…Coupled with NADPH-cytochrome P450 reductase (8), they are assumed to catalyze the terminal hydroxylation of n-alkanes, which represents the first and rate-limiting step in the n-alkane assimilation pathway. The functions of the C. maltosa cytochromes P450 were first apparent from in vitro reconstitution of n-alkane hydroxylase and NADPH-cytochrome P450 reductase purified from n-alkane-grown C. maltosa cells (9) and could be confirmed later by in vivo CO inhibition studies (10). In addition to these investigations, recent studies revealed the enzymatic characters of some P450alk forms by means of heterologous expression in Saccharomyces cerevisiae (11)(12)(13).…”

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confidence: 99%

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Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Ohkuma¹,

Zimmer²,

Iida³

et al. 1998

Journal of Biological Chemistry

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An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway. Eight structurally related P450alk genes have been identified. In the present study, the function of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption. Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain. The strain depleted of all four isoforms could not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation. Growth properties of a series of intermediate disrupted strains, plasmid-based complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

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Section: Discussionsupporting

confidence: 64%

mentioning

confidence: 99%

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Ohkuma¹,

Zimmer²,

Iida³

et al. 1998

Journal of Biological Chemistry

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“…The invertase activity at the cell surface was determined using intact yeast cells after washing with distilled water and resuspending them in MES-buffer. The NADPH-P450 reductase activity was measured as NADPH-cytochrome c reductase activity as described previously (Honeck et al, 1982). The -galactosidase activity was measured in cell-free supernatants according to the procedure described by Rose et al (1990), using an extinction coefficient of 1·91 m 1 cm 1 at pH 7·0.…”

Section: Methodsmentioning

confidence: 99%

Inducible Membranes in Yeast: Relation to the Unfolded-Protein-Response Pathway

Menzel

Vogel

Kärgel

et al. 1997

Yeast

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Overproduction of an endoplasmic reticulum (ER)‐resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the function of the 22 bp unfolded‐protein‐response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded‐protein‐response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high‐level production of the integral membrane protein cytochrome P450 52A3. Moreover, IRE1 gene disruption did not prevent P450‐induced ER proliferation. We suggest that Ire1p‐mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly. © 1997 John Wiley & Sons, Ltd.

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“…The enzyme system is ER membrane-bound and consists of a NADPH-cytochrome P450 reductase transferring electrons, and a cytochrome P450 acting as hydroxylase in a typical monooxygenase reaction. Both enzymes (P450Cml in this case) were first obtained in a highly purified state from alkane-grown C. maltosa EH15 (Riege et al 1981;Honeck et al 1982;Schunck et al 1983a,b), and later from other C. maltosa strains (Avetisova et al 1985;Sokolov et al 1986a,b;Takagi et al 1989;Mauersberger et al 1992a; cf. Table 4).…”

Section: The Role Of Cytochrome P4s0 and Fatty Alcohol Oxidase In Alkmentioning

confidence: 96%

Candida maltosa

Mauersberger

Ohkuma

Schunck

et al. 1996

Nonconventional Yeasts in Biotechnology

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The cytochrome P-450 alkane monooxygenase system of the yeast lodderomyces elongisporus: Purification and some properties of the NADPH-cytochrome P-450 reductase

Cited by 43 publications

References 14 publications

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Inducible Membranes in Yeast: Relation to the Unfolded-Protein-Response Pathway

Candida maltosa

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