Rational re-design of the substrate binding site of flavocytochrome P450 BM3.
Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C(12)-C(20)). Binding or activity with substrates of chain length <C(12) has not been reported. Rational mutagenesis was used to re-design the enzyme to encourage binding of short chain fatty acids (C(4)-C(10)). We show that wild-type P450 BM3 has activity and weak affinity for substrates as short as butyrate (C(4)). However, turnover/binding of short chain substrates is dramatically increased by introducing a novel substrate carboxylate binding site close to the heme. Mutant L181K shows catalytic efficiency (k(cat)/K(M)) increased >13-fold with butyrate, while the L75T/L181K double mutant has k(cat)/K(M) increased >15-fold with hexanoate and binding (K(d)) improved >28-fold for butyrate. Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.
Ost
T W
TW
Department of Chemistry, University of Edinburgh, Edinburgh, UK.
Miles
C S
CS
Murdoch
J
J
Cheung
Y
Y
Reid
G A
GA
Chapman
S K
SK
Munro
A W
AW
D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D016296:Mutagenesis; D009251:NADPH-Ferrihemoprotein Reductase; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; C008757:palmitoleic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases