The role of Thr268 in oxygen activation of cytochrome P450BM-3.
Cytochrome P450BM-3, a catalytically self-sufficient monooxygenase from Bacillus megaterium, catalyzes the omega-n (n = 1-3) hydroxylation of fatty acids in the presence of O2 and NADPH. Like most other P450s, cytochrome P450BM-3 contains a threonine residue (Thr268) in the distal I helix thought to be important for O2 binding and activation. Thr268 has been converted to alanine and the enzymatic properties and heme domain crystal structure determined. Using sodium laurate as the substrate, the mutant exhibited slower rates of O2 and NADPH consumption. In addition, electron transfer is uncoupled from substrate hydroxylation as evidenced by the greater production of water and peroxide in the mutant compared to the wild-type enzyme. The crystal structure of the mutant reveals that the only changes in structure are confined to the site of mutation. These data indicate an important role for Thr268 in O2 binding and activation in the metabolism of sodium laurate by cytochrome P450BM-3.
Yeom
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H
Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana 61801, USA.
Sligar
S G
SG
Li
H
H
Poulos
T L
TL
Fulco
A J
AJ
D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins; D001483:Base Sequence; D003196:Computer Graphics; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013053:Spectrophotometry; D013912:Threonine / Q000737:chemistry*
D001426:Bacterial Proteins; D007850:Lauric Acids; D011994:Recombinant Proteins; C030358:lauric acid; D013912:Threonine; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D010100:Oxygen