Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Abstract

Candida albicans contains ten putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6 × (His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, ω-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (ω-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d 3 -dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the ω-to the (ω-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein.

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…2; Table 8, entries 5, 10 and 14). For ω,ω,ω-trideuterated lauric acid (C12:0) a shift towards (ω-1)-hydroxylation was obtained, and 12-halogenated lauric acids (iodo-, bromo, and chloro-) were converted to 12-oxo-lauric acid via hydrolysis of the oxohalonium species formed by halogen-oxidation [126]. An engineered Candida tropicalis strain was demonstrated to hydroxylate C12:0 (supplied as the corresponding methyl ester) with a productivity of more than 160 g L −1 , and less than 5% formation of the corresponding dicarboxylic acid [130].…”

Self-Sufficient Catalytic System of Human Cytochrome P450 4A11 and NADPH-P450 Reductase

“…Analyses were performed on an Agilent 6890 gas chromatograph coupled to an Agilent 5973 Network Mass Selective Detector in the flame ionization mode as previously described (Kim et al, 2007) …”

“…In addition, it should be noted that covalent heme binding is not a prerequisite for high terminal hydroxylation regiospecificity (Ortiz de Montellano, 2008). Candida albicans CYP52A21, which does not bind its prosthetic heme covalently, cata- lyzes the terminal hydroxylation of lauric acid predominantly (Kim et al, 2007).…”

Fatty Aldehyde Dehydrogenase Multigene Family Involved in the Assimilation of n-Alkanes in Yarrowia lipolytica

“…n-Alkane assimilating yeasts, including Candida tropicalis (8 -10), Candida maltosa (11)(12)(13), Debaryomyces hansenii, Candida albicans (14), Candida bombicola, Candida parapsilosis, Lodderomyces elongisporus, Pichia stipitis, and Yarrowia lipolytica (15)(16)(17), have multiple paralogs of P450ALKs (1). Y. lipolytica, one of the most well studied n-alkane-assimilating yeasts, has 12 genes (ALK1-ALK12) that are predicted to encode P450ALKs (15)(16)(17)(18).…”