CPR determines CYP53A15 product specificity
In the work presented here, we show that the product formed by a given CYP from a specific substrate can vary with the nature of the CPR of the microsomal P450 system that participates in the reaction. When coupled with CPR1, CYP53A15 converts benzoic acid to the expected 4-hydroxybenzoic acid in a single hydroxylation reaction. Similarly, 3-methoxybenzoic acid is demethylated in a single step to 3-hydroxybenzoic acid (Faber et al., 2001; Podobnik et al., 2008). However, when coupled with CPR2, CYP53A15 converts both of these substrates into the same product in a two-step reaction. This product, 3,4-dihydroxybenzoic acid, or protocatechuate, is the phenolic derivative that enters the β-ketoadipate pathway, which is the central pathway for aromatic compound degradation – and benzoic acid detoxification (Harwood and Parales, 1996). This result is of critical importance for biological systems that express more than one functional CPR gene, as it diversifies the metabolic capacity of modular P450 systems.
The step in the pathway that converts 4-hydroxybenzoic acid to protocatechuate is catalysed by 4-hydroxybenzoate-3-monooxygenase, an enzyme that has been extensively studied in several bacteria (Ballou et al., 2005; Palfey and McDonald, 2010). This enzyme is not a cytochrome P450, but belongs to a family of flavin-dependent monooxygenases, which, like CYPs, insert a single atom of molecular oxygen into the substrate and catalyse oxygenation reactions like hydroxylation and epoxidation (Van Berkel et al., 2006). Flavin-dependent monooxygenases use a purely organic cofactor for oxygenation reactions and not a transition metal like the haem in cytochromes P450. These enzymes are relatively abundant in prokaryotes. However, reports on flavin-dependent monooxygenases from fungi are scarce (Kalin et al., 1992; Eppink et al., 2000; Torres Pazmino et al., 2010). Early work on Aspergillus niger identified 3-hydroxybenzoate-4-hydroxylase that hydroxylated 3-hydroxybenzoic acid to protocatechuate, but did not convert 4-hydroxybenzoic acid (Premkumar et al., 1969). The gene encoding this enzyme has not been identified. Therefore, in addition to a hypothetical flavin-dependent monooxygenase catalysing selected steps leading to the formation of protocatechuate, CYP53A15 and the possible combinations of redox partners may be involved in more than one reaction in the pathway of aromatic compound degradation.
Electrostatic binding interactions between basic residues of the cytochrome P450 and clusters of negatively charged acidic residues of CPR are important in the formation and stabilization of P450 redox partner complexes (Paine et al., 2005). Recently, crystallographic and NMR data suggested that CPR exists in equilibrium between physiologically relevant open and closed conformations that differ in the orientation of the FMN-binding domain (Aigrain et al., 2009; Ellis et al., 2009; Hamdane et al., 2009). Electron transfer to the cytochrome P450, or other acceptor, is possible when the reductase is in the open conformation and exposes a new interface on the surface of the FMN-binding domain. Evidence for cooperative substrate binding that we calculated from the rates of ferricyanide reduction agrees with these extensive conformational changes during the CPR catalytic cycle. Multiple redox partner docking sites were identified on the surface of the FMN-binding domain of CPR in helices B, C and F (Shen and Kasper, 1995; Ellis et al., 2009; Jang et al., 2010). Sequence alignment of mammalian and both C. lunatus CPRs reveals that acidic residues involved in redox partner interaction are fairly conserved between human CPR and CPR1 (Fig. S6). Notably, in CPR2 we find several instances where acidic residues are replaced by polar basic (Arg), non-polar basic (Ser) and non-polar hydrophobic (Ala) amino acids. Elimination of these negative charges can alter the mode of interaction between CPR and electron acceptors (Jang et al., 2010), which could also be another explanation for the lower activity of RS2.
Product specificity of the two CPRs can partially be explained by visualizing the 3D complex between CPR1 and CYP53A15 (Fig. 9). Although all the structures were modelled, they help explain the nature of interactions between the two proteins. Molecular dynamics simulations of CYP53A15 show that although the enzyme's haem group is well anchored, it is highly flexible within the active site. The root mean square of the haem movements during the interval of 2.5 ns fluctuates between 0.25 and 0.8 A from its starting position (Fig. S7). It is therefore very likely that the FMN-binding domain of each CPR specifically affects these movements in complex with the CYP, and consequently the activity of each system. We investigated the interaction between CPR1 and CYP53A15 by subjecting the two facing structures to the RosettaDock server that predicted several protein complexes (http://rosettadock.graylab.jhu.edu/). From the predicted low-energy docking structures, we chose the one where the shortest distance between the FMN of CPR1 and the haem of CYP53A15. In the final 2 ns of the CPT-EWALD dynamic simulation, the distance between FMN and the haem iron fluctuated between 8.0 and 10.2 A.
Figure 9. The model of interaction of cytochrome P450 reductase (CPR; yellow) and CYP53A15 (black). The FMN-binding domain of CPR in open conformation interacts with the CYP53A15 that brings the FMN cofactor in close proximity to the CYP haem cofactor and facilitates electron transfer. (NADPH, nicotinamide adenine dinucleotide phosphate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide).
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Consistent with the above, Sevrioukova and Poulos (2010) reported that close contact between P450cam and its redox partner putidaredoxin (Pdx) upon binding enables active site restructuring via haem displacement that couples electron transfer to substrate hydroxylation. Given that the FMN-binding domains of CPR1 and CPR2, share only 32% amino acid identity, it is reasonable to hypothesize that these two domains will modulate the haem group's movements differently when CYP53A15 is complexed with either CPR. Such interactions could result in different product specificities, as the same substrate would assume a slightly altered position in the active site permitting for example, hydroxylation reactions at particular sites of the molecule.
The two CPRs do not greatly affect substrate specificity of CYP53A15, as the enzyme's activity was successfully reconstituted with both redox partners, albeit with different rates of product formation from the same substrates. CYP53A15 reconstituted with CPR1 converted benzoic acid more readily than 3-methoxybenzoic acid. When reconstituted with CPR2, conversion of 3-methoxybenzoic acid was faster than that of benzoic acid. The rates of product formation from both substrates were faster with RS1 compared with RS2.
CPR1 and CPR2 putatively affect the specificity of CYP53A15's detoxification activity
In addition to the differential role endogenous CPRs of C. lunatus play in CYP53A15 product specificity, their physiological function differs as well. Our in vivo experiments that include deletion mutant studies and gene expression data support the putative function of CPR1 in primary metabolism. Combined with the results of in vitro experiments, we suspect that this reductase is the physiological partner of CYP53A15 when the substrate is benzoic acid. The role of CPR2 and its putative function in specialized metabolism is more elusive and seems to be utilized in the metabolism of specific xenobiotic compounds.
While the deletion of cpr1 is not lethal, the growth of the mutant is severely delayed. This phenotype indicates an important role of CPR1 in fungal primary metabolism, specifically as the electron donor to three enzymes in the evolutionary conserved pathway of ergosterol biosynthesis, sterol 22-desaturase (CYP61) and sterol 14α-demethylase (CYP51) and squalene epoxidase (e.g. Tiedje et al., 2007). In fungi, CYP51 is essential and the central drug target of azole-based inhibitors (Kelly et al., 2001). Increased sensitivity to ketoconazole has been observed in the ncp1Δ S. cerevisiae deletion strain (Sutter and Loper, 1989).
Unexpectedly, in the Δcpr1 mutant, CPR2 apparently does not complement the deletion of its paralogue to function as the alternative redox partner. Similar phenotypes have been observed when the homologue of cpr1 was deleted in the fungi Botrytis cinerea and Giberella fujikuroi (Malonek et al., 2004; Siewers et al., 2004). In the latter case, the alternative redox partners were identified to be cytochrome b5 (CYB5) and cytochrome b5 reductase (CBR) (Troncoso et al., 2008). As we have observed, the deletion of the native CPR gene in S. cerevisiae, NCP1, causes poor growth. The double deletion of NCP1 and the gene encoding CBR, CBR1, is lethal (Tiedje et al., 2007). It has also been shown that CYB5 and CBR support CYP51 activity in vitro (Lamb et al., 1999). We therefore conclude that in C. lunatus CPR1 delivers electrons to enzymes in the ergosterol biosynthesis pathway. In the mutant, its role is probably replaced by CYB5 and CBR, but not by CPR2.
Gene expression data show that cpr2 was relatively highly induced by isoeugenol and 3-methoxybenzoic acid, whereas expression of cpr1 was not induced by either of these substrates. Surprisingly, as it does not agree with literature data, expression of neither reductase was induced by benzoic acid (Van Den Brink et al., 2000; Matsuzaki and Wariishi, 2005). Comparison of gene expression of cpr2 relative to cpr1 shows that the fold expression of cpr2 is 10−2 to 10−3 relative to cpr1. The cpr1 gene therefore appears to be constitutively expressed (less inducible) whereas the expression of its more inducible paralogue in non-treated mycelia is lower.
The severe growth inhibition of the Δbph mutant on benzoic acid and the high expression of the coding gene (over 260-fold) indicate that benzoic acid is the main substrate of CYP53. The preferred redox partner in benzoic acid elimination seems to be CPR1, as RS1 has the highest rates of product formation. As well, the benzoic acid-mediated growth inhibition of the Δcpr2 mutant is similar to the wild-type strain that suggests that CPR1, present in both strains, delivers electrons to CYP53A15.
Growth of the wild type is most affected on media supplemented with isoeugenol, and to a lesser extent by 3-methoxybenzoic acid. The inhibition on these two supplemented media is similar in the Δcpr2 and Δbph mutants. As noted above, isoeugenol and 3-methoxybenzoic acid are also the substrates that highly induce cpr2, and do not induce cpr1 gene expression. CPR2 may be the particular CYP53A15 redox partner when substrates are other phenolic compounds particularly those with a 3-methoxy substituent on the aromatic ring. Both RS1 and RS2 convert these phenolic compounds in vitro; however, it appears that these CYP systems (RS1 and RS2 in the wt and RS1 in the Δcpr2 mutant) are not as efficient in eliminating these two inhibitory compounds in vivo. As well, either the product of the reaction, or products of its downstream processing could result in the formation of a toxic intermediate. In the case of 3-methoxybenzoic acid as substrate, the reaction product is protocatechuate. Accumulation of this compound, or its di-oxygenated derivative carboxymuconate was shown to be toxic in bacteria (Parke et al., 2000). When isoeugenol is the substrate, the product of the RS2 is not known. To validate hypotheses of the toxicity of particular intermediates, we would need a better understanding of the sequential enzymatic steps in the pathway of aromatic compound degradation in C. lunatus. The putatively deleterious function of RS2 could also explain the increased synergistic inhibitory effects of benzoic acid with phenolic compounds on the growth of C. lunatus reported previously (Podobnik et al., 2008). If benzoic acid is efficiently eliminated by RS1, the formation of RS2 would lower the amount of available CYP53A15 and perhaps simultaneously produce a toxic intermediate.
While we have identified CPR1 as the preferred redox partner of CYP51 and CYP53A15, the CYPs that prefer CPR2 have yet to be identified. An indication could be the genomic context of these genes in genomes of species of the same taxonomic group. In two Dothideomycete species that possess the cpr2 homologue, a CYP gene of unknown function is in close proximity to the cpr2 gene. In Aspergillus nidulans, this gene belongs to the CYP630 family (Kelly et al., 2009). The cpr1 gene is present in all (12) analysed Dothideomycete genomes and has no neighbouring CYP genes.
CYP53A15 has multiple functions in primary and secondary metabolism
CYP53 enzymes in fungi use benzoic acid as the substrate to produce 4-hydroxybenzoic acid and are essential in detoxifying aromatic compounds (Fukuda et al., 1996; Faber et al., 2001; Fraser et al., 2002; Matsuzaki and Wariishi, 2005; Podobnik et al., 2008). CYP53s can have other substrates, and these can vary between fungal species. For example, 3-methoxybenzoic is demethylated to 3-hydroxybenzoic acid in C. lunatus and A. niger, but not in Phanerochaete chrysosporium (Faber et al., 2001; Matsuzaki and Wariishi, 2005). Although reports on substrate specificity can vary due to differences in experimental set-up and substrates tested, substrate structural requirements are generally rather strict. Mono-substitutions on the phenyl ring are allowed only at certain positions with specific groups, while the carboxyl group positions the substrate into the active site so that a spectral shift is induced which helps to identify substrates spectrophotometrically (Faber et al., 2001; Podobnik et al., 2008). With our flourometric assay, we identified additional compounds without the carboxyl group, like phenylacetaldehyde and cinnamic acid, as CYP53A15 substrates. A broader than expected spectrum of possible substrates in turn extends the putative physiological roles of CYP53A15.
To study the CYP system in vivo, we expressed the redox partners, as well as the entire monooxygenase system in the S. cerevisiae with its native CPR gene (NCP1) knocked out. We followed growth on control and benzoic acid-supplemented media. As well, we have analysed the ergosterol content of transformants. As noted above, the ncp1Δ strain grows poorly. The wild-type growth is recovered after complementation with the native reductase, or C. lunatus CPR2 or RS2. Perhaps CPR2 better complements the deletion because of putatively closer evolutionary relationships between the CPR2 family of Pezizomycotina and the CPR family of ascomycetous yeasts (Saccharomycotina) (Lah et al., 2008). In contrast, the complementation with CPR1 did not restore the wild-type growth. In these transformants, the measured levels of membrane ergosterol content agree with the observed phenotype, where poor growth is associated with lower ergosterol levels caused by defects in its biosynthetic pathway (Venkateswarlu et al., 1998).
Complementation of the ncp1Δ strain with CYP53A15, or RS1 from C. lunatus resulted in no improvement of growth and almost no growth respectively. However, the ergosterol levels in these transformants with poor growth on solid media or liquid culture were relatively high. It has been observed that although ergosterol levels in the ncp1Δ mutant are about 25% of those in the wt, this was still the predominant sterol found in membranes (Venkateswarlu et al., 1998). Despite the fact that a strong correlation exists between ergosterol content and fungal dry mass, the amount of ergosterol in fungal tissue is not constant (Parsi and Gorecki, 2006; Silva et al., 2010; and references therein). It depends among others on the fungal species, developmental stage and growth conditions (growth media, pH and temperature).
We have shown in vitro that phenylacetaldehyde, an intermediate of phenylalanine metabolism, can be a substrate of CYP53A15. As well, it has been reported that bph gene expression was induced by L-phenylalanine (Fujii et al., 1997). Based on our results and relevant literature reports, it is therefore possible that CYP53A15 (when not complemented with CPR2) assumes a function in yeast metabolism that does not impede ergosterol biosynthesis but still retards growth. CYP53A15 putatively associates with the alternative redox partners, CYB5 and CBR, as has been observed in fungi, insects and plants (e.g. Brankova et al., 2007; Murataliev et al., 2008; Troncoso et al., 2008). In double transformants, RS1 and RS2, it may associate with CPR1 and CPR2, respectively, to mediate different reaction outcomes. These outcomes, in turn, may cause the observed differences in phenotypes of transformed strains.