cytochrome-c-t and resorufin

Anchorage of recombinant proteins onto the outer membrane of gram-negative bacteria is an attractive solution for protein library screening and whole cell biocatalysis if a membrane environment is required or mass transfer into the cell is limiting. Autotransporters have been successfully applied for surface display of various heterologous proteins. Still, many underlying parameters for achieving active enzymes are not known. Here, we systematically tested different linkers between passenger and the membrane embedded β-barrel of the autotransporter. The linker can have influence on aspects such as steric orientation of the passenger, distance to the outer membrane and accessibility of active sites. Six linker variants for display of the cytochrome P450 reductase were tested. Cytochrome c reduction by the cytochrome P450 reductase varied fivefold and was highest by introduction of a flexible glycine-serine region. When these variants were co-expressed with surface displayed CYP1A2, product concentration for paracetamol differed between 0.22 μM and 2.5 μM and for resorufin between 0.23 μM to 1 μM. The best glycine/serine containing sequence, that turned out to be best for CPR display, was then introduced into the linker for displaying CYP1A2. In comparison, up to 7.9 μM paracetamol and up to 1.69 μM resorufin were obtained with this new variant. The differences were not caused by changes in the number of displayed enzymes. To our knowledge, this is the first systematic study on engineering the linker for surface display of recombinant enzymes.

Topics: Acetaminophen; Adhesins, Escherichia coli; Amino Acid Motifs; Binding Sites; Catalytic Domain; Cell Membrane; Cloning, Molecular; Cytochrome P-450 CYP1A2; Cytochromes c; Escherichia coli; Gene Expression; Genetic Engineering; Kinetics; Models, Molecular; Oxazines; Plasmids; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Fusion Proteins; Substrate Specificity

The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP).. The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin.. The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate.. Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.

Topics: Animals; Antioxidants; Benzoflavones; beta-Naphthoflavone; Carbon Monoxide; Cytochrome P-450 Enzyme System; Cytochromes b5; Cytochromes c; Ferricyanides; Flavonoids; Fluorometry; Male; Microsomes; NADPH-Ferrihemoprotein Reductase; Oxazines; Oxidation-Reduction; Rabbits; Spectrophotometry; Thioctic Acid; Time Factors