Probing the molecular determinants of coenzyme selectivity in the P450 BM3 FAD/NADPH domain.
Bacillus megaterium P450 BM3 (BM3) is an NAD(P)H-binding diflavin reductase exhibiting substantial coenzyme specificity for NADPH over NADH. The side chains of serine 965, arginine 966 and lysine 972 in its FAD-binding domain bind the NADPH 2'-phosphate. Optical, kinetic and thermodynamic properties of S965A, R966A and K972A FAD domains were analyzed singly and combined with the FAD-shielding W1046A mutation. Steady-state and stopped-flow kinetic studies demonstrated substantially decreased NADPH affinity versus wild-type (WT) FAD domain (146-fold for the S965A K(d)). Considerable catalytic efficiency increases (the ratio of specificity constants, k(cat)/K(m), for the coenzymes) with NADH were observed for each point mutant over WT (570-fold in K972A), along with increased rates of NADH-dependent FAD reduction (k(lim) elevated 5.2-fold in R966A). In combination with W1046A, considerable (37 to 56-fold) improvements over WT were seen in the k(lim) parameters with NADH for all double mutants. Each 2'-phosphate binding point mutant produced large increases in FAD potential (111 mV in R966A), despite large distances between these residues and the FAD isoalloxazine ring (18-21 A), suggesting long range conformational influences on FAD environment. The W1046A/K972A mutant abolished NADPH selectivity (8340-fold coenzyme selectivity switch towards NADH), with ramifications for BM3's biotechnological exploitation using the cheaper NADH coenzyme.
Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester, UK. adrian.dunford@Manchester.ac.uk
Adrian J
AJ
Dunford
Hazel M
HM
Girvan
Nigel S
NS
Scrutton
Andrew W
AW
Munro
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism
D001426:Bacterial Proteins; D005182:Flavin-Adenine Dinucleotide; D012694:Serine; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D008239:Lysine