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Genetic diversity of cytochrome P450 1A2 with different metabolic activities in domestic cats.

ABSTRACT: Knowledge of genetic polymorphisms of metabolizing enzymes of medical drugs and xenobiotics including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP1A2, one of the major CYP isoforms in the feline liver. Wild-type and three non-synonymous polymorphic variants, but no synonymous variant, were identified in feline CYP1A2 in 50 alleles of domestic cats in Japan. Metabolic parameters, Km and Vmax, of ethoxyresorufin hydroxylation by CYP1A2 were shown to range within two times for identified non-synonymous variants by using a heterologous coexpression system. The results confirmed the polymorphic nature of CYP1A2 as a basis for effective application of medicines and prevention of adverse reactions in the treatment of domestic cats as well as for hereditary disorders.

SUBMITTER: Sugiyama S

PROVIDER: S-EPMC6656809 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Genetic diversity of cytochrome P450 1A2 with different metabolic activities in domestic cats.

Sugiyama Souta S Uno Yasuhiro Y Amano Tomoko T Kitazawa Takio T Teraoka Hiroki H

The Journal of veterinary medical science 20190521 7

Knowledge of genetic polymorphisms of metabolizing enzymes of medical drugs and xenobiotics including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP1A2, one of the major CYP isoforms in the feline liver. Wild-type and three non-synonymous polymorphic variants, but no synonymous variant, were identified in feline CYP1A2 in 50 alleles of domestic cats in Japan. Metabolic parameters, Km and Vmax, of ethoxyresorufin hydroxylation by CYP1A2 were shown to range wit ...[more]

PMID: 31118351

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Genetic diversity of cytochrome P450 2A with different metabolic activities in domestic cats.

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Investigation of the major cytochrome P450 1A2 genetic variant in a healthy Tibetan population in China.

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Cytochrome P450 1A2 Is Incapable of Oxidizing Bilirubin Under Physiological Conditions.

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| S-EPMC6813656 | biostudies-literature

Co-expression of active human cytochrome P450 1A2 and cytochrome P450 reductase on the cell surface of Escherichia coli.

Project description:BackgroundHuman cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug-drug interactions, drug clearance and activation of prodrugs. Their biocatalytic behavior is a key parameter during drug development which requires preparative synthesis of CYP related drug metabolites. However, recombinant expression of CYP enzymes is a challenging bottleneck for drug metabolite biosynthesis. Therefore, we developed a novel approach by displaying human cytochrome P450 1A2 (CYP1A2) and cytochrome P450 reductase (CPR) on the surface of Escherichia coli.ResultsTo present human CYP1A2 and CPR on the surface, we employed autodisplay. Both enzymes were displayed on the surface which was demonstrated by protease and antibody accessibility tests. CPR activity was first confirmed with the protein substrate cytochrome c. Cells co-expressing CYP1A2 and CPR were capable of catalyzing the conversion of the known CYP1A2 substrates 7-ethoxyresorufin, phenacetin and the artificial substrate luciferin-MultiCYP, which would not have been possible without interaction of both enzymes. Biocatalytic activity was strongly influenced by the composition of the growth medium. Addition of 5-aminolevulinic acid was necessary to obtain a fully active whole cell biocatalyst and was superior to the addition of heme.ConclusionWe demonstrated that CYP1A2 and CPR can be co-expressed catalytically active on the cell surface of E. coli. It is a promising step towards pharmaceutical applications such as the synthesis of drug metabolites.

| S-EPMC4736170 | biostudies-literature

Significant increase in phenacetin oxidation on L382V substitution in human cytochrome P450 1A2.

Project description:Human CYP1A2 is an important drug-metabolizing enzyme, similar in sequence to CYP1A1 but with distinct substrate specificity. We have previously shown that residue 382 affected CYP1A1 and CYP1A2 specificities with alkoxyresorufins. To determine whether this residue is also important for the metabolism of other substrates, we have investigated phenacetin oxidation by single (T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/N312L) mutants of CYP1A2. The enzymes were expressed in Escherichia coli and purified. All the CYP1A2 mutants that contained the L382V substitution displayed much higher activities than the wild-type enzyme, with k(cat) values 3-fold higher, in contrast to other mutants, for which k(cat) decreased. Likewise, a significant increase in specificity, expressed as the k(cat)/K(m) ratio, was observed for the mutants containing the L382V substitution. The efficiency of coupling of reducing equivalents to acetaminophen formation was decreased for all the single mutants except L382V, for which the coupling increased. This effect was also observed with multiple CYP1A2 mutants containing the L382V substitution. Low activities of the four other single mutants were likely caused by dramatically increased uncoupling to water. In contrast, the increase in activity of the L382V-containing mutants resulted from decreased water formation. This finding is consistent with molecular dynamics results, which showed decreased phenacetin mobility leading to increased product formation. The results of these studies confirm the importance of residue 382 in CYP1A2-catalyzed oxidations and show that a single residue substitution can dramatically affect enzymatic activity.

| S-EPMC2908983 | biostudies-literature

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