The Sulfolobus solfataricus electron donor partners of thermophilic CYP119: an unusual non-NAD(P)H-dependent cytochrome P450 system.
CYP119 from Sulfolobus solfataricus is the first well-characterized thermophilic cytochrome P450 enzyme. The endogenous substrate for this enzyme is not known but it hydroxylates lauric acid in a reaction supported by surrogate mesophilic electron donors. However, reconstitution of a high-temperature catalytic system requires identification of the normal thermophilic electron donor partners of CYP119. Here, we describe cloning, expression in Escherichia coli, and characterization of the requisite electron donor partners from S. solfataricus. One is a thermostable ferredoxin and the second a 2-oxoacid-ferredoxin oxidoreductase that utilizes pyruvic acid rather than NAD(P)H as the source of reducing equivalents. CYP119 is the only cytochrome P450 to date known to obtain electrons from a non-NAD(P)H-dependent protein. The two thermophilic partners have been used to reconstitute a catalytic system that hydroxylates lauric acid at 70 degrees C, and the optimal conditions for this system have been defined. This first high-temperature in vitro catalytic system represents an important step in the development of industrially relevant catalysts.
Puchkaev
Andrei V
AV
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA 94143-2280, USA.
Ortiz de Montellano
Paul R
PR
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics; D004579:Electron Transport; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019766:Genes, Archaeal; D006358:Hot Temperature; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D005288:Ferredoxins; D007850:Lauric Acids; D011994:Recombinant Proteins; C030358:lauric acid; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus; D007658:Ketone Oxidoreductases; C023096:2-oxoglutarate synthase