Catalytically functional flavocytochrome chimeras of P450 BM3 and nitric oxide synthase.
P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains. Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras. Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified. All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity. However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case. The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties.
Fuziwara
Shigeyoshu
S
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan.
Sagami
Ikuko
I
Rozhkova
Elena
E
Craig
Daniel
D
Noble
Michael A
MA
Munro
Andrew W
AW
Chapman
Stephen K
SK
Shimizu
Toru
T
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D005420:Flavoproteins / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013698:Templates, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D005420:Flavoproteins; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D002118:Calcium