Heterologous coexpression of the benzoate-para-hydroxylase CYP53B1 with different cytochrome P450 reductases in various yeasts.

Cytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large-scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self-sufficient P450s for their potential as whole-cell biocatalysts. Most P450s are, however, not self-sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450-reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450-CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad-range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole-cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450-based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole-cell biocatalyst was emphasized, supporting earlier findings.

A benzoate-activated promoter from Aspergillus niger and regulation of its activity.

The filamentous fungus Aspergillus niger is able to use benzoic acid as a sole carbon source by conversion to protocatechuic acid and subsequent metabolism. Synthesis of the first enzyme in this metabolic pathway, benzoate p-hydroxylase, is encoded by the bphA gene and positively regulated at the transcriptional level by benzoic acid. Methyl benzoate and para-aminobenzoate also act as inducers of the bphA gene. We show that bphA expression in A. niger in response to benzoate is confined to a 530-bp fragment from the bphA promoter region from -787 to -509 bp from the transcriptional start site. Electrophoretic mobility-shift assays show that a benzoate-response element, consisting of a single 6-bp sequence (5'-TAGTCA-3') within a 51-bp sequence in this region, is most likely to be involved in binding of one or more proteins that modulate the activity of the promoter in response to benzoic acid. We show through fusion of promoter fragments with the green fluorescent protein that the active sequences are located within a 200-bp sequence containing the TAGTCA benzoate-response element. Identification of the benzoate-response element in the bphA promoter region constitutes the first step in the development of a benzoate-inducible promoter system that could be used to control gene expression in fungi, and possibly in other organisms, such as plant and animal cells.

Antifungal activity of cinnamic acid derivatives involves inhibition of benzoate 4-hydroxylase (CYP53).

AIMS: CYP53A15, from the sorghum pathogen Cochliobolus lunatus, is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes. METHODS AND RESULTS: In our work, we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus and two other fungi, Aspergillus niger and Pleurotus ostreatus. To elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15. CONCLUSION: We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development.

Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase.

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

CYP53A15 of Cochliobolus lunatus, a target for natural antifungal compounds.

A novel cytochrome P450, CYP53A15, was identified in the pathogenic filamentous ascomycete Cochliobolus lunatus. The protein, classified into the CYP53 family, was capable of para hydroxylation of benzoate. Benzoate is a key intermediate in the metabolism of aromatic compounds in fungi and yet basically toxic to the organism. To guide functional analyses, protein structure was predicted by homology modeling. Since many naturally occurring antifungal phenolic compounds are structurally similar to CYP53A15 substrates, we tested their putative binding into the active site of CYP53A15. Some of these compounds inhibited CYP53A15. Increased antifungal activity was observed when tested in the presence of benzoate. Some results suggest that CYP53A15 O-demethylation activity is important in detoxification of other antifungal substances. With the design of potent inhibitors, CYP53 enzymes could serve as alternative antifungal drug targets.

Heterologous expression of the benzoate para-hydroxylase encoding gene (CYP53B1) from Rhodotorula minuta by Yarrowia lipolytica.

There is currently an increasing number of cytochrome P450 (CYP450) monooxygenase encoding genes becoming available from various genome-sequencing projects. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. In this study, the CYP53B1 gene, which encodes a benzoate para-hydroxylase, was successfully cloned from Rhodotorula minuta and overexpressed in Yarrowia lipolytica E150. Multiple copies of the CYP53B1 cDNA were cloned under the POX2 promoter, while the Y. lipolytica CPR was cloned under the isocitrate lyase promoter. Whole cell biotransformation of benzoic acid to para-hydroxybenzoic acid (pHBA) was used to analyse the hydroxylase activity of the recombinant Y. lipolytica UOFS Y-2366. Different induction conditions were tested in shake flask cultures. The highest concentration of pHBA produced by UOFS Y-2366 was 1.6 g l(-1) after 200 h when stearic acid was repeatedly added to the media. R. minuta accumulated up to 1.8 g l(-1) of pHBA within only 24 h. Thus, the specific hydroxylase activity of Y. lipolytica UOFS Y-2366 [approximately 0.07 U (g dry wt.)(-1)] was about 30 times lower than the specific hydroxylase activity of R. minuta [2.62 U (g dry wt.)(-1)]. However, the hydroxylation activity obtained with Y. lipolytica was one of the highest hydroxylation activities thus reported for whole cell biotransformation studies carried out with yeasts expressing foreign CYP450s.

Purification and characterization of benzoate-para-hydroxylase, a cytochrome P450 (CYP53A1), from Aspergillus niger.

Benzoate-para-hydroxylase (CYP51A or BpH) and NADPH:cytochrome P450 reductase from the filamentous fungus Aspergillus niger were purified to apparent homogeneity, using an overproducing A. niger strain. This is the first membrane-bound fungal cytochrome P450 to be isolated and characterized. Combining BpH with NADPH:cytochrome P450 oxidoreductase in the presence of the phospholipid dilauryl phosphatidylcholine restored the BpH activity, although to only a minor extent. Spectral analysis of BpH showed characteristic spectra for a cytochrome P450. Substrate binding studies with purified BpH as a function of temperature and as a function of pH were performed. Temperature-dependent studies, at pH 8.0, showed that the simplified spin equilibrium model originally proposed for camphor binding to cytochrome P450cam (M. T. Fisher and S. G. Sligar, 1987, Biochemistry 26, 4797-4803) also applies to the benzoate-BpH system. Two equilibrium constants were determined, K(1) for substrate binding without a spin change and K(2) for the spin change of the benzoate-BpH complex. pH-dependent binding studies showed that both K(1) and K(2) increase with pH, indicative of a higher affinity. As K(1) decreases more strongly with pH than K(2), we suggest that benzoate first binds to a binding site on the outside of the protein in a pH-dependent way, followed by transfer to the inside of the protein causing a spin change at the heme iron. The strong pH dependence of K(1) could be the result of the need to break salt bridges at the binding site on the outside of the protein. pH-dependent kinetic studies with microsomes showed that the apparent K(M) values followed the trend observed for benzoate binding to purified BpH, while k(cat) values were virtually constant between pH 6.6 and 8.0 and decreased above pH 8, probably due to loss of productive interaction between BpH and NADPH:cytochrome P450 oxidoreductase. Research into the substrate specificity of BpH showed that BpH can only use benzoic acid and some of its derivatives. Monosubstitution on the phenyl ring is allowed but only at certain positions with specific, not too large groups. Substitution always leads to a lower affinity of the substrate. With one exception, all substrates were converted to their 4-hydroxy derivative. The exception, 3-methoxybenzoate, was demethylated to yield 3-hydroxybenzoate only. The restricted number of substrates and the specificity in catalysis suggest that BpH is not a general-purpose hydroxylase but that its role is confined to benzoate hydroxylation in the beta-ketoadipate pathway of A. niger.

Regulation of expression of the Aspergillus niger benzoate para-hydroxylase cytochrome P450 system.

Cytochrome P450 enzyme systems are found throughout nature and are involved in many different, often complex, bioconversions. In the endoplasmic reticulum of the filamentous fungus Aspergillus niger a cytochrome P450 enzyme system is present that is capable of the para-hydroxylation of benzoate. The expression of the two genes encoding the components of this system, the cytochrome P450 gene encoding benzoate para-hydroxylase (bphA) and the gene encoding cytochrome P450 reductase (cprA), is inducible by benzoate. The BPH system was used as a model system to study the mechanisms that result in co-regulation of both components of an eukaryote cytochrome P450 enzyme system. Deletion analysis of the transcription control regions of cprA and bphA resulted in the identification of a region that was involved in benzoate induction of gene expression. The functional competence of the cprA Benzoate Responsive Region thus defined was demonstrated directly by cloning this fragment upstream of a constitutively expressed mini-promoter and analysing expression of the hybrid transcription control region in a lacZ reporter system. Further analysis of cprA gene expression revealed a clear quantitative discrepancy between induction at the protein level (approximately 4-fold) and at the transcription level (> 20-fold). The majority of the transcripts observed after benzoate induction (cprAbeta) were larger then the constitutively expressed cprAalpha transcript. The difference in size between the cprAalpha and cprAbeta transcript is caused by differential promoter use. As the longer cprAbeta transcript carries a small uORF we propose that post-transcriptional regulation of CPR expression underlies the discrepancy in the degree of induction at the protein and transcriptional level. Our results show that regulation of CPR expression is particularly complex, involving regulatory promoter elements, differential promoter use and regulation at the post-transcriptional level.

Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate.

Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine

Cytochrome P450rm from Rhodotorula minuta catalyzes 4-hydroxylation of benzoate.

Rhodotorula minuta, a red yeast, produces a cytochrome P450, tentatively named P450rm, catalyzing the formation of isobutene from isovalerate. We found that P450rm interacted with benzoate and the dissociation constant of P450rm for benzoate was 36 microM. A reconstituted system that consisted of purified P450rm and cytochrome P450 reductase catalyzed the 4-hydroxylation of benzoate in addition to the formation of isobutene; the turnover rate was approximately 40 nmol/min/nmol P450rm. The P450rm-monooxygenase system was specific for benzoate and did not catalyze hydroxylation of other aromatic carboxylates. Since only a benzoate 4-hydroxylase that requires tetrahydropteridine has been isolated to date, P450rm appears to be the first isolated cytochrome P450 that acts as a benzoate 4-hydroxylase. The P450rm-monooxygenase system in microsomes of R. minuta might function in the degradation of L-phenylalanine on the pathway to beta-ketoadipate.

The mobile flavin of 4-OH benzoate hydroxylase.

Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function.

This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.

Specific immuno-detection of benzoate-para-hydroxylase with antibodies raised against synthetic peptides.

As a model system for the industrial use of fungal cells in the enzymatic conversion of chemicals, the parahydroxylation of benzoate was studied. To increase the amount of benzoate-para-hydroxylase (BPH, EC 1.14.13.12.) in the cell the gene coding for the enzyme (bphA) was cloned and expressed in Aspergillus niger. Detection of the enzymatic activity of the protein was not reproducible. It was decided to raise an antiserum for immuno-detection purposes. Sufficient benzoate-para-hydroxylase for immunization could not be obtained; therefore the synthetic-peptide strategy was used. We demonstrate that synthesis of antigenic determinants, can be useful in the production of highly specific reagents for the detection of proteins. The availability of monospecific polyclonal sera opens new possibilities in functional studies and purification of benzoate-para-hydroxylase.