Isozyme function of n-alkane-inducible cytochromes P450 in Candida maltosa revealed by sequential gene disruption

Item Type:	Article
Title:	Isozyme function of n-alkane-inducible cytochromes P450 in Candida maltosa revealed by sequential gene disruption
Creators Name:	Ohkuma, M. and Zimmer, T. and Iida, T. and Schunck, W.H. and Ohta, A. and Takagi, M.
Abstract:	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.
Keywords:	Alkanes, Candida, Cytochrome P-450 Enzyme System, Enzyme Induction, Fungal Genes, Fungal Proteins, Gene Deletion, Genetic Complementation Test, Genetic Markers, Hydroxylation, Isoenzymes, Substrate Specificity, Animals, Rats
Source:	Journal of Biological Chemistry
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Volume:	273
Number:	7
Page Range:	3948-3953
Date:	13 February 1998
Official Publication:	https://doi.org/10.1074/jbc.273.7.3948
PubMed:	View item in PubMed

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