ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.     

Linkage of cation binding and folding in human telomeric quadruplex DNA

Formation of DNA quadruplexes requires monovalent cation binding. To characterize the cation binding stoichiometry and linkage between binding and folding, we carried out KCl titrations of Tel22 (d[A(GGGTTA)₃]), a model of the human telomere sequence, using a fluorescent indicator to determine [K⁺]_free and circular dichroism to assess the extent of folding. At [K⁺]_free = 5 mM (sufficient for > 95% folding), the apparent binding stoichiometry is 3K⁺/Tel22; at [K⁺]_free = 20 mM, it increased to 8-10K⁺/Tel22. Thermodynamic analysis shows that at [K⁺]_free = 5 mM, K⁺ binding contributes approximately − 4.9 kcal/mol for folding Tel22. The overall folding free energy is − 2.4 kcal/mol, indicating that there are energetically unfavorable contributions to folding. Thus, quadruplex folding is driven almost entirely by the energy of cation binding with little or no contribution from other weak molecular interactions.     

Functional polymorphisms in the CYP3A4, CYP3A5, and CYP21A2 genes in the risk for hypertension in pregnancy

An intronic single nucleotide polymorphism (SNP) in the CYP3A5 gene (CYP3A5∗3; SNP rs776746) affects RNA splicing and enzymatic activity. The CYP3A5∗3 frequency increased with distance from the equator and natural selection has been proposed to explain the worldwide distribution of this allele. CYP3A activity has been related with the risk for hypertension in pregnancy, a major cause of morbidity and mortality among women, and CYP3A5∗3 could reduce the risk for this disease in populations from regions with high sodium and water availability. The CYP3A5 genotype was related with blood pressure in the general population, but the effect on the risk for hypertension in pregnancy has not been evaluated.We compared the allele and genotype frequencies of three functional SNPs in the CYP3A5 (rs776746), CYP3A4 (rs2740574), and CYP21A2 (rs6471) genes between pregnant women who developed hypertension (n = 250) or who remained normotensive (control group, n = 250). In addition, we sequenced the full CYP3A5 coding sequence in 40 women from the two groups to determine whether some gene variants could explain the risk for hypertensive pregnancies in our population.Allele and genotype frequencies did not differ between hypertensive and normotensive women for the three CYP variants. We did not find CYP3A5 nucleotide changes that could explain a higher risk for hypertension in pregnancy. Our data suggests that the variation in CYP3A5, CYP3A4, and CYP21A2 did not contribute to the risk for hypertension in pregnancy in our population.     

Inhibitory effect of nitric oxide on the induction of cytochrome P450 3A4 mRNA by 1,25-dihydroxyvitamin D3 in Caco-2 cells

Cytochrome P450 (CYP)-dependent drug metabolism decreases in vivo and in cultured hepatocytes under various immunostimulatory conditions. Nitric oxide (NO) released during inflammation is presumed to be involved in this phenomenon. CYP3A4, which is abundant in the liver and small intestine and participates in the metabolism of various drugs, is known to be induced by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in the colon carcinoma cell line Caco-2. In this study we examined whether NO affected CYP3A4 gene expression induced by 1,25(OH)₂D₃ in Caco-2 cells. Induction of CYP3A4 mRNA by 1,25(OH)₂D₃ was suppressed in a dose-dependent manner by treatment with the NO donors NOR-4 (15–500 μM) or S-nitroso-N-acetyl-penicillamine (30 μM-1 mM), which spontaneously release NO. These results indicated that NO has an inhibitory effect on the induction of CYP3A4 mRNA by 1,25(OH)₂D₃ in Caco-2 cells. Treatment with the guanylate cyclase inhibitor ODQ failed to prevent the inhibition of induction of CYP3A4 mRNA by 1,25(OH)₂D₃. 8-Bromo cGMP had no effect on 1,25-(OH)₂D₃-induced CYP3A4 gene expression. Therefore, the suppression of CYP3A4 mRNA by NO might be mediated through a guanylate cyclase-independent pathway.     

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K_S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.     

Comparison of warfarin sensitivity between rat and bird species

Scattering coumarin derivative rodenticides in broad areas have caused primary- and secondary-poisoning incidents in non-target wild birds. In this study, we compared factors determining warfarin sensitivity between bird species and rats based on vitamin K 2,3-epoxide reductase (VKOR) kinetics, VKOR inhibition by warfarin and warfarin metabolism assays. In VKOR characterization, chickens and ostriches showed significantly lower enzymatic efficiencies than rats (one-sixth and one-third, respectively), suggesting bird species depend more on a non-VKOR vitamin K source. On the other hand, the inhibition constants (K_i) of VKOR for warfarin were significantly different between chickens and ostriches (11.3 ± 2.5 μM and 0.64 ± 0.39 μM, respectively). Interestingly, the ostrich K_i was similar to the values for rats (0.28 ± 0.09 μM). The K_i results reveal a surprising possibility that VKOR in some bird species are easily inhibited by warfarin. Warfarin metabolism assays also showed a large inter-species difference in bird species. Chickens and ostriches showed higher metabolic activity than that of rats, while mallards and owls showed only a slight ability to metabolize warfarin. In this study, we clarified the wide inter-species difference that exists among birds in xenobiotic metabolism and sensitivity to a rodenticide.     

Beta sheet 2–alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.     

Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy

Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (K_m = 4.63 ± 0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4–1.2 pH units, likely due to repositioning of the cofactor and modification of the pK_a of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3–3, 26–58 and 124–568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The K_m^l-Cth of E333 substitution variants is increased ~ 17-fold, while K_m^l-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (k_cat/K_m^l-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (k_cat/K_m^l-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (k_cat/K_m^l-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.     

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ± 4 μM, K_Mg^2 + = 294 ± 22 μM, K_L-gln = 11 ± 1 mM, and k_cat = 2.2 ± 0.2 s^− 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed

Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is a major threat to malaria control programmes. Cytochome P450-mediated detoxification is an important resistance mechanism. CYP6M2 is over-expressed in wild populations of permethrin resistant A. gambiae but its role in detoxification is not clear. CYP6M2 was expressed in Escherichia coli and a structural model was produced to examine its role in pyrethroid metabolism. Both permethrin and deltamethrin were metabolized. Rates were enhanced by A. gambiae cytochrome b₅ with kinetic parameters of K_M = 11 ± 1 ??M and k_cat = 6.1 ± 0.4 per min for permethrin (1:1 cis-trans) and K_M = 2.0 ± 0.3 ??M and k_cat = 1.2 ± 0.1 per min for deltamethrin. Mass spectrometry and NMR analysis identified 4′-hydroxy deltamethrin and hydroxymethyl deltamethrin as major and minor deltamethrin metabolites respectively. Secondary breakdown products included cyano(3-hydroxyphenyl)methyl deltamethrate and deltamethric acid. CYP6M2 was most highly transcribed in the midgut and Malpighian tubules of adult A. gambiae, consistent with a role in detoxification. Our data indicates that CYP6M2 plays an important role in metabolic resistance to pyrethroids and thus an important target for the design of new tools to combat malaria.     

Spectral resolution of a second binding site for Nile Red on cytochrome P4503A4

Nile Red is sequentially metabolized by cytochrome P4503A4 to the N-monoethyl and N-desethyl products, which typifies the metabolism of many amine containing drugs. Sequential metabolism of a single substrate results in complex kinetics that confound predictive models of drug clearance. As a fluorescent model for drugs which undergo sequential metabolism, Nile Red provides the opportunity to monitor drug-CYP interactions wherein the fluorescent properties of Nile Red could, in principle, be exploited to determine individual rate and equilibrium constants for the individual reactions. Previously, it was shown that Nile Red binds at the active site and fluoresces (K_D ≈ 50 nM) with maximum emission at ∼620 nm, but it was unclear whether a red-shifted emission, at ∼660 nm, consisted of only free Nile Red or Nile Red bound at a second site on the protein. Here, equilibrium binding studies, including ‘reverse titrations’ spanning low ratios of CYP3A4/Nile Red, indicate two binding sites for Nile Red with a contribution to the ‘red emission’ greater than can be accounted for by free Nile Red. Singular value decomposition affords basis spectra for both spectral components and fits well to the experimentally determined concentration dependence of Nile Red emission. In addition, the red spectral component, with an apparent K_D = 2.2 μM, is selectively eliminated by titration with the known allosteric effectors of CYP3A4, α-napthoflavone and testosterone. Furthermore, the double mutant L2311F/D214E, previously demonstrated to perturb a peripheral allosteric site, lacks the red-emitting Nile Red binding site, but retains the blue-emitting site. Together these data indicate that a second Nile Red site competes with other effectors of CYP3A4 at a site that results in Nile Red emission at 660 nm.     

Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana

Formation of the methylenedioxy bridge is an integral step in the biosynthesis of benzo[c]phenanthridine and protoberberine alkaloids in the Papaveraceae family of plants. This reaction in plants is catalyzed by cytochrome P450-dependent enzymes. Two cDNAs that encode cytochrome P450 enzymes belonging to the CYP719 family were identified upon interrogation of an EST dataset prepared from 2-month-old plantlets of the Mexican prickly poppy Argemone mexicana that accumulated the benzo[c]phenanthridine alkaloid sanguinarine and the protoberberine alkaloid berberine. CYP719A13 and CYP719A14 are 58% identical to each other and 77% and 60% identical, respectively, to stylopine synthase CYP719A2 of benzo[c]phenanthridine biosynthesis in Eschscholzia californica. Functional heterologous expression of CYP719A14 and CYP719A13 in Spodoptera frugiperda Sf9 cells produced recombinant enzymes that catalyzed the formation of the methylenedioxy bridge of (S)-cheilanthifoline from (S)-scoulerine and of (S)-stylopine from (S)-cheilanthifoline, respectively. Twenty-seven potential substrates were tested with each enzyme. Whereas CYP719A14 transformed only (S)-scoulerine to (S)-cheilanthifoline (K_m 1.9 ± 0.3; k_cat/K_m 1.7), CYP719A13 converted (S)-tetrahydrocolumbamine to (S)-canadine (K_m 2.7 ± 1.3; k_cat/K_m 12.8), (S)-cheilanthifoline to (S)-stylopine (K_m 5.2 ± 3.0; k_cat/K_m 2.6) and (S)-scoulerine to (S)-nandinine (K_m 8.1 ± 1.9; k_cat/K_m 0.7). These results indicate that although CYP719A14 participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine and berberine formation in A. mexicana.