Probing the molecular determinants of coenzyme selectivity in the P450 BM3 FAD/NADPH domain

Adrian J. Dunford, Hazel M. Girvan, Nigel S. Scrutton, Andrew W. Munro

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)


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 Kd). Considerable catalytic efficiency increases (the ratio of specificity constants, kcat/Km, 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 (klim elevated 5.2-fold in R966A). In combination with W1046A, considerable (37 to 56-fold) improvements over WT were seen in the klim 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 Å), 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.

Original languageEnglish
Pages (from-to)1181-1189
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Issue number8
Early online date1 Apr 2009
Publication statusPublished - 1 Aug 2009
Externally publishedYes


Dive into the research topics of 'Probing the molecular determinants of coenzyme selectivity in the P450 BM3 FAD/NADPH domain'. Together they form a unique fingerprint.

Cite this