- Formate Oxidase (FOx) from Aspergillus oryzae: One Catalyst Enables Diverse H2O2-Dependent Biocatalytic Oxidation Reactions
-
An increasing number of biocatalytic oxidation reactions rely on H2O2 as a clean oxidant. The poor robustness of most enzymes towards H2O2, however, necessitates more efficient systems for in situ H2O2 generation. In analogy to the well-known formate dehydrogenase to promote NADH-dependent reactions, we here propose employing formate oxidase (FOx) to promote H2O2-dependent enzymatic oxidation reactions. Even under non-optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved.
- Tieves, Florian,Willot, Sébastien Jean-Paul,van Schie, Morten Martinus Cornelis Harald,Rauch, Marine Charlène Renée,Younes, Sabry Hamdy Hamed,Zhang, Wuyuan,Dong, JiaJia,Gomez de Santos, Patricia,Robbins, John Mick,Bommarius, Bettina,Alcalde, Miguel,Bommarius, Andreas Sebastian,Hollmann, Frank
-
-
Read Online
- Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19
-
Toxic and pharmacokinetic profiles of drug candidates are evaluated in vivo often using monkeys as experimental animals, and the data obtained are extrapolated to humans. Well understanding physiological properties, including drug-metabolizing enzymes, of monkeys should increase the accuracy of the extrapolation. The present study was performed to compare regio- and stereoselectivity in the oxidation of propranolol (PL), a chiral substrate, by cytochrome P450 2D (CYP2D) enzymes among humans, cynomolgus monkeys and marmosets. Complimentary DNAs encoding human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19 were cloned, and their proteins expressed in a yeast cell expression system. The regio- and stereoselective oxidation of PL enantiomers by yeast cell microsomal fractions were compared. In terms of efficiency of expression in the system, the holo-proteins ranked CYP2D6 ≒ CYP2D17 CYP2D19. This may be caused by the bulky side chain of the amino acid residue at position 119 (leucine for CYP2D19 vs. valine for CYP2D6 and CYP2D17), which can disturb the incorporation of the heme moiety into the active-site cavity. PL enantiomers were oxidized by all of the enzymes mainly into 4-hydroxyproranolol (4-OH-PL), followed by 5-OH-PL and N-desisopropylpropranolol (NDP). In the kinetic analysis, apparent Km values were commonly in the μM range and substrate enantioselectivity of R-PL m and Vmax values for the formation of the three metabolites from PL enantiomers. The activity to produce NDP tended to be higher for the monkey enzymes, particularly CYP2D17, than for the human enzyme. These results indicate that in the oxidation of PL enantiomers by CYP2D enzymes, stereoselectivity is similar but regioselectivity is different between humans and monkeys.
- Narimatsu, Shizuo,Nakata, Toshiyuki,Shimizudani, Takeshi,Nagaoka, Kenjiro,Nakura, Hironori,Masuda, Kazufumi,Katsu, Takashi,Koeda, Akiko,Naito, Shinsaku,Yamano, Shigeru,Miyata, Atsuro,Hanioka, Nobumitsu
-
experimental part
p. 146 - 152
(2012/01/07)
-
- Regioselective preparation of 5-hydroxypropranolol and 4′-hydroxydiclofenac with a fungal peroxygenase
-
An extracellular peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4′-hydroxydiclofenac (4′-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4′-OHD in yields up to 20% and 65%, respectively. 18O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4′-OHD originated from H2O2, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.
- Kinne, Matthias,Poraj-Kobielska, Marzena,Aranda, Elisabet,Ullrich, Rene,Hammel, Kenneth E.,Scheibner, Katrin,Hofrichter, Martin
-
body text
p. 3085 - 3087
(2010/01/16)
-
- Product inhibition and dose-dependent bioavailability of propranolol in the isolated perfused rat liver preparation
-
We investigated in the isolated perfused rat liver (IPRL) whether product inhibition of metabolism contributes to the dose-dependent bioavailability of propranolol, a drug with a high, but saturable, hepatic first-pass effect. (±)-Propranolol was infused in the IPRL, using a recirculating design, for three 36-min periods (n = 9). Mean steady-state reservoir, i.e. hepatic inflow concentrations (C(in)), were 4.97, 10.4, and 20.4 μM, respectively. Mean reservoir concentrations of the metabolites 4'-hydroxypropranolol, 5'- hydroxypropranolol, N-desisopropylpropranolol, and naphthoxylactic acid (NLA), a major side-chain-oxidation metabolite, increased disproportionately with propranolol dose, but their production rate did not reach steady state. In separate experiments (n = 4), perfusate containing 7.1, 12.8, and 21.6 μM (±)-propranolol, corresponding to administration rates of 114, 205, and 346 nmol/min, respectively, was passed through the liver for 30 min each using a single-pass design. The bioavailability (hepatic outflow concentration/C(in)) of propranolol increased with C(in) from 0.012 to 0.150 to 0.288 in the recirculating IPRL. In the single-pass IPRL the increase (0.0077 in 0.0669 to 0.136) was significantly less (P 0.001). The greater bioavailability of propranolol in recirculating experiments was attributed to product inhibition since metabolites do not accumulate with the single-pass design. NLA did not appear to be the inhibiting metabolite because in further single-pass experiments with propranolol C(in) of 21.6 μM the presence of NLA (21.6 μM) in perfusate had no effect on propranolol bioavailability (n = 7) compared with control experiments (n = 5). These data suggest that, with the recirculating IPRL, dose-dependent bioavailability of propranolol is due to competitive inhibition of propranolol metabolism by propranolol metabolites, which is distinct from the noncompetitive product inhibition that has been reported to accompany chronic propranolol administration.
- Ghabrial,Nand,Stead,Smallwood,Morgan
-
p. 931 - 936
(2007/10/02)
-