Unique properties of purified, Escherichia coli-expressed constitutive cytochrome P4504A5.
Cytochromes P450 of the 4A family metabolize a variety of fatty acids, prostaglandins, and eicosanoids mainly at the terminal carbon (omega-hydroxylation) and, to a lesser extent, at the penultimate carbon [(omega-1)-hydroxylation]. In the present study, cytochrome P4504A5 (4A5) has been successfully expressed in Escherichia coli, with an average yield of enzyme of approximately 80 nmol/liter of cells. Spectroscopic characterization of the purified enzyme, using electron paramagnetic resonance and absolute and substrate-perturbed optical difference spectroscopy, showed that the heme of resting 4A5 is primarily low spin, but is converted primarily to high spin by substrate binding. The kcat and Km values for laurate omega-hydroxylation were 41 min-1 and 8.5 microM, respectively, in the absence of cytochrome b5, and 138 min-1 and 38 microM, respectively, in the presence of cytochrome b5. Hydroxylation of palmitate was dependent on the presence of cytochrome b5; kcat and Km values were 48 min-1 and 122 microM, respectively. Hydroxylation of arachidonic acid was barely detectable and was unchanged by the addition of cytochrome b5.
Hosny
G
G
Department of Biochemistry, The University of Texas Health Science Center at San Antonio 78284-7760, USA.
Roman
L J
LJ
Mostafa
M H
MH
Masters
B S
BS
D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors / Q000138:chemical synthesis; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity / Q000235:genetics
D007527:Isoenzymes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A