Minipig cytochrome P450 3A, 2A and 2C enzymes have similar properties to human analogs

BACKGROUND: The search for an optimal experimental model in pharmacology is recently focused on (mini)pigs as they seem not only to be an alternative source of cells and tissues for xenotherapy but also an alternative species for studies on drug metabolism in man due to similarities between (mini) pig and human drug metabolizing systems. The purpose of this work is to characterize minipig liver microsomal cytochromes P450 (CYPs) by comparing their N-terminal sequences with corresponding human orthologs. RESULTS: The microsomal CYPs exhibit similar activities to their human orthologous enzymes (CYP3A4, nifedipine oxidation; 2A6, coumarin 7-hydroxylation; 2D6, bufuralol 1'-hydroxylation; 2E1, p-nitrophenol hydroxylation; and 2C9, tolbutamide hydroxylation). Specific minipig CYP (2A, 2C and 3A) enzymes were partially purified and proteins identified by immunostaining (using antibodies against the respective human CYPs) were used for N-terminal amino acid sequencing. From comparisons, it can be concluded that the sequence of the first 20 amino acids at the N-terminus of minipig CYP2A is highly similar to human CYP2A6 (70% identity). The N-terminal sequence of CYP2C shared about 50% similarity with human 2C9. The results on the minipig liver microsomal CYP3A yielded identical data with those obtained for amino acid sequences of the pig CYP3A29 showing 60% identity with human CYP3A4. CONCLUSIONS: Thus, our results further support the view that minipigs may serve as model animals in pharmacological/toxicological studies with substrates of human CYP enzymes, namely, of the CYP3A and CYP2A forms

Highly Purified Liver Microsomal Cytochrome P450: Properties and Catalytic Mechanism

Recent studies in this laboratory on two forms of cytochrome P450 purified to homogeneity from rabbit liver microsomes are reviewed. The two forms, phenobarbital-inducible P450LM2 and 5,6-benzoflavone-inducible P450LM4, differ in subunit molecular weight, identity of the C-terminal amino acid, optical and EPR spectra, and other properties. As isolated, oxidized P450LM2 is in the low spin state, whereas P450LM4 is largely, but non entirely, in the high spin state. Mechanistic studies have shown the following: (a) P450LM2 may accept two electrons, calculated per heme, from dithionite or NADPH in the presence of catalytic amounts of the reductase, and may donate two electrons to various oxidizing agents, including molecular oxygen. (b) Hydrogen peroxide is formed in the reconstituted system in the presence of NADPH and oxygen, and the amount varies with the substrate added. (c) Hydrogen peroxide and other hydroperoxides apparently donate the oxygen atom inserted into substrate during hydroxylation in the absence of 0 2 and an external donor. (d) Stopped flow spectrophotometry has provided evidence for two distinct oxygenated complexes of the reduced cytochrome. The reductase and cytochrome b5 may play an effector role in increasing the rate of decomposition of the second complex during oxygen insertion into substrate. A scheme is proposed for the mechanism of action of purified P450LM2, based on these and other findings

Highly Purified Liver Microsomal Cytochrome P450: Properties and Catalytic Mechanism

Recent studies in this laboratory on two forms of cytochrome P450 purified to homogeneity from rabbit liver microsomes are reviewed. The two forms, phenobarbital-inducible P450LM2 and 5,6-benzoflavone-inducible P450LM4, differ in subunit molecular weight, identity of the C-terminal amino acid, optical and EPR spectra, and other properties. As isolated, oxidized P450LM2 is in the low spin state, whereas P450LM4 is largely, but non entirely, in the high spin state. Mechanistic studies have shown the following: (a) P450LM2 may accept two electrons, calculated per heme, from dithionite or NADPH in the presence of catalytic amounts of the reductase, and may donate two electrons to various oxidizing agents, including molecular oxygen. (b) Hydrogen peroxide is formed in the reconstituted system in the presence of NADPH and oxygen, and the amount varies with the substrate added. (c) Hydrogen peroxide and other hydroperoxides apparently donate the oxygen atom inserted into substrate during hydroxylation in the absence of 0 2 and an external donor. (d) Stopped flow spectrophotometry has provided evidence for two distinct oxygenated complexes of the reduced cytochrome. The reductase and cytochrome b5 may play an effector role in increasing the rate of decomposition of the second complex during oxygen insertion into substrate. A scheme is proposed for the mechanism of action of purified P450LM2, based on these and other findings

Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase iota

DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn2+ than Mg2+. The human germline R96G variant impairs both Mn2+-dependent and Mg2+-dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg2+-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1-445) proteins. The presence of Mn2+ (0.15 mM) instead of Mg2+ (2 mM) caused a 770-fold increase in efficiency (kpol/Kd,dCTP) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in Kd,dCTP. The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in kpol/Kd,dCTP for dCTP insertion opposite G with Mg2+ when compared with wild type, substantially attenuated by substitution with Mn2+. Crystal structures of pol ι ternary complexes, including the primer terminus 3′-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg2+ or Mn2+, revealed that Mn2+ achieves more optimal octahedral coordination geometry than Mg2+, with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.1