Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

Abstract

Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

Natural Product Diversification by One‐Step Biocatalysis using Human P450 3A4

“…[98] Several recombinant enzymes were established for the preparative synthesis of these human demethylation metabolites. [99][100][101][102] The question remains why the products 17 and 19 were yet unknown as human metabolites of 4, although they appeared as major products of the P450 3A4 biocatalysis in this study. The benzylic position 10 of 4 is readily oxidised [103] and N-oxidation is a frequent transformation carried out by human P450 3A4.…”

“…GC-MS confirmed the mass and the HSQC spectrum provided 13 C shifts of the product. No literature data could be found as a reference for 5hydroxypiperitone in research databases trans-6-hydroxypiperitone ( (6), 135 (12), 126 (59), 111 (35), 98 (100), 69 (41), 55 (18), 41 (29). By comparing to literature 1 H NMR data of both trans-and cis-6-hydroxypiperitone isomers, which show different chemical shifts due to the hydroxyl group sitting either in a pseudoaxial (trans to isopropyl) or -equatorial (cis to isopropyl) position, the formed product structure was suggested to be trans-6-hydroxypiperitone.…”

“…[143,144] (+)-nootkatone ( (75), 79 (69), 67 (25), 53 (25), 41 (50). 8 Hz, 9-H), 2.64 (1H, dddd, J = 12.7, 12.6 3.2, 2.7 Hz, 7-H), 2.24 (1H, dd, J = 16.9, 14.0 Hz, 3α-H), 2.12 (1H, dd, J = 16.9, 4.0 Hz, 3β-H), 1.92-1.85 (2H, m, 4-, 8α-H), 1.84 (1H, ddd, J = 13.1, 3.1, 3.0 Hz, 6α-H), 1.62 (3H, br, 13-H), 1.37 (1H, ddd, J = 13.1, 12.9, 3.0, 8β-H), 1.17 (3H, br, 15-H), 0.95 (1H, dd, J = 12.9, 12.8 Hz, 6β-H), 0.82 (3H, d, J = 6.8 Hz, 14-H); GC-MS (EI + , 70 eV): m/z (%) = 234 ([M] + , 25), 216 (88), 191 (100), 174 (31), 166 (31), 159 (31), 145 (38), 137 (50), 131 (31), 121 (38), 107 (38), 91 (38), 67 (38), 55 (25), 41 (44). -H), 1.92 (1H, ddd, J = 12.6, 3.1, 2.7 Hz, 6α-H), 1.63 (1H, ddd, J = 12.8, 3.2, 3.0 Hz, 7-H), 1.34 (1H, ddd, J = 12.5, 12.3, 4.9 Hz, 8β-H), 1.25 (3H, d, J = 0.4 Hz, 13-H), 1.06 (3H, s, 15-H), 1.00-0.95 (1H, m, 6β-H), 0.97 (3H, d, J = 6.8 Hz, 14-H); GC-MS (EI + , 70 eV): m/z (%) = 234 ([M] + , 5), 216 (60), 206 (51), 201 (25), 191 (14), 177 (100), 161 (95), 147 (43), 134 (69), 119 (48), 105 (65), 91 (65), 79 (48), 67 (22), 55 (31), 41 (43).…”

“…The chemical shifts were almost identical to those in the literature. [82,83] (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9-hydroxynootkatone (13, C 15 H 22 O 3 , yellow oil, 6 mg, 4 %): 1 H NMR (300 MHz, CDCl 3 ): δ = 5.83 ChemCatChem (1H, br, 1-H), 4.40 (1H, dd, J = 2.9, 2.9 Hz, 9-H), 2.69 (1H, dd, J = 4.6, 0.3 Hz, 12-H), 2.59 (1H, d, J = 4.6 Hz, 12-H), 2.35 (1H, d, J = 17.9, 13.6 Hz, 3α-H), 2.27 (1H, dd, J = 17.9, 4.5, 0.7 Hz, 3β-H), 2.17 (1H, ddd, J = 12.8, 3.4, 3.0 Hz, 7-H), 2.09-2.01 (1H, m, 8α-H), 2.00-1.88 (2H, m, 4-, 6α-H), 1.48 (1H, J = 12.8, 12.7, 3.2 Hz, 8β-H), 1.27 (3H, s, 15-H), 1.25 (3H, s, 13-H), 0.97 (1H, J = 12.9, 12.7 Hz, 6β-H), 0.95 (3H, d, J = 6.8 Hz, 14-H); GC-MS (EI + , 70 eV): m/z (%) = 250 ([M] + , 22), 232 (22), 217 (44), 191 (100), 175 (44), 161 (44), 147 (56), 137 (56), 121 (94), 105 (78), 91 (78), 79 (61), 69 (61), 55 (66), 41 (83). The absolute configuration was assigned by comparison to literature data, which were almost identical.…”

Roles of Ferredoxin-NADP+ Oxidoreductase and Flavodoxin in NAD(P)H-Dependent Electron Transfer Systems

“…The cindoxin reductase displays strong similarity to NADPH-dependent ferredoxin reductases, and cindoxin reductase is replaced by Escherichia coli flavodoxin reductase. Klenk et al [ 128 ] reported two novel cyt P450s in Arthrobacter sp. , and they proposed the potential cluster in the genome including two cyt P450s and electron partners flavodoxin and flavodoxin reductase.…”