Functional expression and characterization of cytochrome P450 52A21 from Candida albicans.
Candida albicans contains 10 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 6x(His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-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 omega- to the (omega-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.
Kim
Donghak
D
Department of Pharmaceutical Chemistry, University of California, San Francisco, Genentech Hall, N572D, 600 16th Street, San Francisco, CA 94158-2517, USA.
Cryle
Max J
MJ
De Voss
James J
JJ
Ortiz de Montellano
Paul R
PR
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System