Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase
ABSTRACT
Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.
INTRODUCTION
Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus and is the causative pathogen of Septoria leaf blotch in wheat crops. This is the most common cereal crop disease in Europe and can result in devastating losses in crop yield (1, 2). Currently the most widely used fungicides for the control of M. graminicola are demethylase inhibitors (DMIs), which bind to the target cytochrome P450 enzyme, CYP51 (ERG11).
CYP51s are ubiquitous in nature and mediate the essential sterol 14α-demethylation step in the sterol biosynthetic pathway (3). The 14α-demethylated products are precursors leading to formation of cholesterol (mammals) and ergosterol (fungi) and the formation of a variety of 24-alkylated and desaturated sterols in algae, plants, and protozoa (3). Cholesterol, ergosterol, and sitosterols (plants) play an important structural role in regulating membrane fluidity and permeability of plasma membranes and indirectly modulate the distribution and activity of membrane proteins and ion channels (3, 4). In addition, sterols are precursors of steroid hormones in mammals, brassinosteroids in plants, and ecdysteroids in insects. In yeast and fungi, the CYP51 enzymes are synonymous with lanosterol and eburicol 14α-demethylation in the production of ergosterol, but as this study shows, M. graminicola CYP51 is able to demethylate only eburicol, in the presence of its native reductase partner, exhibiting novel substrate specificity.
The introduction of new azole antifungal compounds has allowed control of M. graminicola infections in wheat to be maintained despite increased tolerance/resistance. The most recently introduced azole is the triazolinethione derivative prothioconazole. However, the control of this disease has been threatened by the identification of mutations in the CYP51 enzyme that are recognized for being involved in M. graminicola populations developing resistance to these fungicides (5, 6). Similar mutations in the CYP51 enzyme have also been observed in the clinical setting with Candida albicans and are responsible for azole-resistant infections in patients (7, 8). Therefore, understanding the biochemical nature of the CYP51 enzyme from M. graminicola and its interactions with azole antifungal drugs is paramount to agricultural economics and food security.
NADPH cytochrome P450 reductase (CPR) is the main redox partner for CYP51 (and other eukaryotic cytochromes P450) and is essential for functional metabolism. CPR is a flavoprotein containing equal amounts of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), each localized within its own structural domain joined together by an α-helical peptide bridge region. CPR catalyzes the transfer of two electrons from exogenous NADPH to the FAD prosthetic group and then to the FMN prosthetic group before donating electrons in two discrete one-electron steps to the CYP acceptor (9). This is known as the cytochrome P450 catalytic cycle. Most eukaryotes, including M. graminicola, contain only one CPR gene, which must be able to interact with and reduce the wide and divergent range of CYPs present within each organism.
In this study, we have cloned, expressed, and purified the CPR enzyme from M. graminicola (MgCPR) and have successfully reconstituted the CPR enzyme with the native CYP51 enzyme from M. graminicola in order to catalyze the 14α-demethylation of eburicol. In contrast to other fungal CYP51 proteins studied so far, we demonstrate specificity for eburicol in the reaction, and we speculate on the reason for the lack of activity with the substrate lanosterol. In addition, we have demonstrated the effectiveness of several agricultural azole fungicides at inhibiting the CYP51 reaction catalyzed by the MgCPR/MgCYP51 redox pairing, thus producing a functional method to evaluate the effects of potential new DMIs on MgCYP51.
MATERIALS AND METHODS
Chemicals.
Growth media, ampicillin, and isopropyl-β-d-thiogalactopyranoside (IPTG) were purchased from Formedium, Ltd. (Hunstanton, United Kingdom). Chemicals used in the preparation of phosphate buffers were purchased from Fisher Scientific (Loughborough, United Kingdom). Eburicol was synthesized by David Nes (Texas Tech University, USA). Ni2+-nitrilotriacetic acid (NTA) agarose was obtained from Qiagen (Crawley, United Kingdom). All other chemicals were purchased from Sigma (Poole, United Kingdom), unless otherwise stated.
Sequence alignment of CPR proteins.
An alignment of 12 selected CPR protein sequences was constructed using ClustalX version 1.8 (http://www.clustal.org/). The CPR sequences used were those of Aspergillus fumigatus isoenzyme 1 (AfCPR1) (UniProtKB accession number Q4WM67), Penicillium digitatum (PdCPR) (K9G4M4), Trichophyton rubrum (TrCPR) (F2SI13), Mycosphaerella graminicola (MgCPR) (F9XJP5), Botryotinia fuckeliana (BfCPR) (M7UV93), Blumeria graminis (BgCPR) (N1JBN9), Saccharomyces cerevisiae (ScCPR) (P16603), Candida albicans (CaCPR) (C4YHD6), Aspergillus fumigatus isoenzyme 2 (AfCPR2) (Q4X224), Phanerochaete chrysosporium (PcCPR) (Q9HDG2), Homo sapiens (HsCPR) (P16435), and Rattus norvegicus (RnCPR) (P00388). BLAST2 (http://blast.ncbi.nlm.nih.gov/) was used to calculate percent sequence identities between CPR proteins.
Heterologous expression, purification, and characterization of MgCPR.
The M. graminicola CPR gene (MgCPR) sequence was derived from the M. graminicola genome database (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html/), taking the DNA sequence from chromosome 9, bases 1621899 to 1623695, and synthesized by Eurofins MWG Operon (Ebersberg, Germany) with the nucleotide sequence optimized for expression in Escherichia coli. The gene was designed to be flanked by NdeI and HindIII restriction sites at the 5′ and 3′ ends, respectively, for cloning and with a C-terminal 6-histidine tag for purification. To aid overexpression, the first eight amino acids were altered to MALLLAVF as described by Barnes et al. (10). MgCPR was cloned into the pCWori+ expression vector and transformed into E. coli DH5α cells. Overnight cultures were used to inoculate Terrific broth containing ampicillin (100 μg ml−1 final concentration) and grown at 37°C and 170 rpm for 6 h. IPTG (1 mM) and riboflavin (10 mg liter−1) were added, and expression was carried out at 25°C and 160 rpm for 42 h. MgCPR protein was isolated as described by Arase et al. (11), with the exception that the sonication buffer was modified to contain 2% (wt/vol) sodium cholate and no Tween 20. The solubilized protein was purified using Ni2+-NTA agarose and eluted in 0.1 M potassium phosphate (pH 6.8) containing 25% (wt/vol) glycerol and 1% (wt/vol) histidine. The purified MgCPR protein was dialyzed overnight at 4°C to remove the histidine. SDS-polyacrylamide gel electrophoresis was undertaken to assess protein purity, with staining with Coomassie brilliant blue R-250. The absolute spectrum of the purified MgCPR protein was recorded between 300 and 700 nm on a Hitachi U-3310 UV-visible spectrophotometer (San Jose, CA). The extinction coefficient 21.4 mM cm−1 (12) was used to calculate the concentration of MgCPR. The NADPH-reduced absorption spectrum of MgCPR was determined after the addition of 10 mM NADPH.
Kinetic characterization of MgCPR.
The ability of the CPR to catalyze the reduction of cytochrome c was calculated by measuring the change in absorbance at 550 nm when cytochrome c is reduced and applying the extinction coefficient of 21.1 mM−1 cm−1 (13). The assay system contained an NADPH regeneration system consisting of 0.2 M Tris-HCl (pH 7.8), 2.5 mM glucose-6-phosphate, and 3 U ml−1 glucose-6-phosphate dehydrogenase. A final concentration of 1 nM MgCPR was added to the assay mixture. Substrate saturation experiments were performed by varying the cytochrome c concentration at 50 μM NADPH and by varying the NADPH concentration at 100 μM cytochrome c. The reference cuvettes (path length, 10 mm) contained the same assay mixture except that the NADPH was replaced by an equivalent volume of 0.2% (wt/vol) sodium bicarbonate. The total volume of the assay was 1 ml. Km values were determined by nonlinear regression using the Michaelis-Menten equation. Analyses were performed using ProFit 5.01 (QuantumSoft, Zurich, Switzerland).
CYP51 reconstitution assays.
The CYP51 reconstitution assay system used was based on that of Lepesheva et al. (14) and contained 1 μM C. albicans CYP51 (15) or M. graminicola CYP51 (16), 2 μM MgCPR, 60 μM lanosterol or 60 μM eburicol, 50 μM dilaurylphosphatidylcholine (DLPC), 4% (wt/vol) 2-hydroxypropyl-β-cyclodextrin (HPCD), 0.4 mg ml−1 isocitrate dehydrogenase, 25 mM trisodium isocitrate, 50 mM NaCl, 5 mM MgCl2, and 40 mM MOPS (pH ∼7.2). Assay mixtures were incubated at room temperature (22°C) or 37°C for 5 min prior to initiation with 4 mM β-NADPH-Na4, followed by shaking for 30 min at 37°C or 2 h at 22°C. Sterol metabolites were recovered by extraction with ethyl acetate and dried in a vacuum centrifuge. Samples were derivatized using N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and tetramethylsilane (TMS) in pyridine before analysis by gas chromatography-mass spectrometry (GC-MS) (17).
Km determination for eburicol with MgCYP51 was performed using the same reconstitution assay system, except the concentration of eburicol was varied from 5 to 150 μM. All determinations were undertaken in triplicate.
Determinations of 50% inhibitory concentrations (IC50s) were performed using the MgCPR/MgCYP51 reconstitution assay system with eburicol as described above. Agricultural azole fungicides were added in 2.5 μl dimethylformamide, followed by 15 min of incubation with shaking at 22°C prior to assay initiation with 4 mM β-NADPH-Na4. Samples were incubated for a further 2 h with shaking at 22°C prior to termination and extraction with ethyl acetate. The IC50 in this study is defined as the inhibitor concentration required to cause a 50% inhibition of CYP51 activity under the stated assay conditions.
Homology modeling and ligand docking.
Structural modeling of wild-type Mycosphaerella graminicola CYP51 was undertaken using an automated homology modeling platform as previously described (18). The Protein Data Bank (PDB) templates adopted by the pipeline were as follows: 3DBG (chain B), Streptomyces coelicolor CYP170A1; 4LXJ (chain A) Saccharomyces cerevisiae CYP51 lanosterol 14-alpha demethylase with lanosterol bound; 3L4D chain D (crystal structure of sterol 14-alpha demethylase [CYP51] from Leishmania infantum in complex with fluconazole); 4G3J chain D (sterol 14-alpha demethylase [CYP51] from Trypanosoma brucei); 3MDM chain A (cytochrome P450 46A1 [Homo sapiens]); and 2CIB (CYP51 from Mycobacterium tuberculosis). Lanosterol and eburicol ligands were docked by superposition upon the heme-lanosterol positions of the structure of Saccharomyces cerevisiae CYP51 (PDB 4LXJ) (19). The coordinates for eburicol were obtained from PubChem (CID 65172).
RESULTS
The MgCPR sequence contains conserved FMN, FAD, and NADPH binding domains found in other CPR sequences.
MgCPR shared greatest sequence identity with CPR enzymes from the higher fungi B. fuckeliana (71%), A. fumigatus AfCPR1 (70%), P. digitatum (68%), T. rubrum (68%), and B. graminis (60%), whereas sequence identities with the yeasts C. albicans and S. cerevisiae were only 45% and 43%. MgCPR shared only 43% sequence identity with the CPR of the basidiomycete filamentous fungus P. chrysosporium. Therefore, significant sequence divergence exists among fungal CPR proteins. Surprisingly, A. fumigatus CPR isoenzyme 2 shared only 38% sequence identity with MgCPR, similar to that with the human (39%) and rat (38%) CPR enzymes, suggesting that AfCPR2 may fulfill a different biochemical role in vivo than AfCPR1. An alignment of these 12 CPR proteins (Fig. 1) indicated that conservation among the FMN, FAD, and NADPH binding regions (20) was variable. The FAD-2 binding region was the most highly conserved, with 7 out of 10 residues being conserved, and the FAD-3 binding region was the least conserved (1 out of 5 residues). Also of note are the six-amino-acid conserved region of the FMN-1 (SQxGTAE) binding region and the five sequential conserved residues in the FMN-2 (TYGEG), FMN-3 (FGLGN), and FAD-2 (RYYSI) binding regions. Purified MgCPR exhibits characteristic CPR spectra. Overexpression of MgCPR in E. coli resulted in protein yields of ∼80 nmol per liter of culture. SDS-polyacrylamide gel electrophoresis confirmed that MgCPR had an apparent molecular mass of 80 kDa, which was close to the predicted value of 78,256 Da (including the six-histidine C-terminal extension), and was over 90% pure as judged by staining intensity with Coomassie brilliant blue R-250.
FIG 1
CPR enzymes have characteristic absorbance spectra related to their FAD and FMN prosthetic groups. The absolute spectrum of purified MgCPR (Fig. 2) showed maximal absorption at 454 nm and 380 nm, which was typical for a native CPR protein (21, 22). The addition of NADPH to the protein (followed by air reoxidation) resulted in a decrease in absorption at 454 nm and the appearance of an additional absorption peak at 585 nm, which is characteristic of the air-stable neutral flavin semiquinone (21).
FIG 2
MgCPR catalyzes the reduction of cytochrome c.
The ability of MgCPR to reduce cytochrome c was assessed and measured to enable comparison with other CPR enzymes. The change in rate of the initial velocity of cytochrome c reduction in different concentrations of cytochrome c and NADPH was measured, and turnover rates of the two substrates (NADPH and oxidized cytochrome c) to their products (NADP and reduced cytochrome c) were calculated. The substrate velocity curves for NADPH and cytochrome c (Fig. 3) were best fitted using the Michaelis-Menten equation. The Km for cytochrome c was 10.8 ± 1 μM, and that for NADPH was 6.5 ± 0.3 μM. The maximum turnover observed was 95 ± 7 s−1 in the presence of 120 μM NADPH and 100 μM cytochrome c.
FIG 3
MgCPR functions as a redox partner for CYP51.
MgCPR was reconstituted with C. albicans CYP51 (CaCYP51) and M. graminicola CYP51 (MgCYP51) to evaluate the ability of MgCPR to donate electrons to cytochrome P450 enzymes. In this system, CaCYP51 demethylated both eburicol (0.37 min−1) and lanosterol (0.28 min−1), confirming that MgCPR was fully functional. However, no demethylation was seen with either lanosterol or eburicol when MgCPR was used in conjunction with the native redox partner MgCYP51 under the same conditions (37°C, 30 min). Reducing the MgCPR/MgCYP51 incubation temperature to 22°C resulted in the demethylation of eburicol (0.14 min−1) but not lanosterol. Increasing the duration of the MgCPR/MgCYP51 reaction from 30 min to 2 h increased the demethylated eburicol product from 7% (0.14 min−1) to 25.5% (0.13 min−1), but MgCYP51 still did not metabolize lanosterol (Fig. 4). We conclude that MgCYP51 is an eburicol-dependent CYP51 enzyme.
FIG 4
Substrate inhibition of MgCYP51.
The eburicol velocity curve for MgCYP51 at 22°C showed clear evidence of substrate inhibition (Fig. 5). This suggests that eburicol binds to at least two different sites on the MgCYP51 protein forming distinct substrate and inhibitor complexes. Previous eburicol binding studies with MgCYP51 protein (16) showed Michaelis-Menten kinetics with no evidence for allosterism (Hill number close to one), suggesting that the second inhibitory eburicol binding site is distant relative to the normal substrate binding site. Further studies are required to discern the mechanism of eburicol inhibition of the MgCYP51 reaction.
FIG 5
Eburicol neatly docks in the heme cavity.
Examination of the yeast CYP51 structure (Fig. 6A) reveals 14 residues within 3.5 Å of bound lanosterol: Y126, T130, Y140, F236, F241, G130, L380, H381, S382, L383, F384, F506, S508, and M509. When the modeled Mycosphaerella graminicola CYP51 structure is superimposed, the heme cavity structure is broadly similar, with 11 equivalent residues found to be within 3.5Å of bound lanosterol; Y123, Y137, F233, F238, A311, I381, H382, S383, Y519, S521, and L522. However, analysis of spatial overlaps shows that four residues in MgCYP51 possess conflicts with the bound position of lanosterol (Fig. 6B). One of the most notable overlaps is with I381, where both the CD1 and CG2 atoms (0.864 Å and 0.766 Å, respectively) are found to overlap the third and fourth sterol rings of lanosterol, around positions 14 and 18. These projected overlaps in the simulated docking of lanosterol indicate that it cannot be docked in the same sterol substrate docking orientation relative to the heme group and residues bordering the cavity as in Saccharomyces cerevisiae CYP51. In contrast, for the simulated docking of eburicol (Fig. 6C), there are no such conflicts. Eburicol is neatly accommodated within the heme cavity in a way similar to that for lanosterol in Saccharomyces cerevisiae CYP51, with no spatial overlaps identified with I381. No specific hydrogen bonds between eburicol and residues bordering the cavity were identified, suggesting weaker longer-range van der Waals interactions and steric restriction within the cavity as being predominantly responsible for the selectivity shown.
FIG 6
MgCYP51 activity is inhibited by azoles.
IC50 determinations using 1 μM MgCYP51 and 2 μM MgCPR and the fungicides epoxiconazole, tebuconazole and triadimenol confirmed the tight binding shown by Parker et al. (16). All three agricultural fungicides caused inhibition of CYP51 activity, resulting in IC50s of ∼0.8, ∼1.25, and ∼1.4 μM, respectively (Fig. 7). Prothioconazole showed no significant inhibition at concentrations up to 145 μM, concurring with the weak, atypical binding seen by Parker et al. (16). However, the desthio form of prothioconazole used in this study strongly inhibited MgCYP51, resulting in an IC50 of ∼0.6 μM.
FIG 7
DISCUSSION
Septoria leaf blotch is a major cause of economic losses in agriculture due to fungal infection and is controlled by azole fungicides, more recently the triazolinethione derivative prothioconazole. Controlling this disease is, therefore, crucial for food security. Azole antifungal drugs cause accumulation of 14-methylated sterols and cell growth inhibition, with a block to ergosterol production. Interestingly, the major sterol accumulating during treatment is eburicol (6). This is consistent with the novel observation in this study that eburicol is the substrate of choice for MgCYP51; usually fungal CYP51s can also metabolize lanosterol. Eburicol is produced by 24-methylation of lanosterol, which is the preferred substrate for 24-methyl transferase, so this is presumably required in the natural pathway, leading to the production of ergosterol in M. graminicola. The overexpression of the MgCPR protein described here has allowed a functional reconstitution system to be developed in which eburicol was a prerequisite, and it leads the way for the use of pathogen-relevant assays for fungicide discovery.
The successful overexpression of MgCPR in E. coli resulted in a protein yield that was half the 160 nmol liter−1 obtained for C. albicans CPR (20) and 10-fold less than the 840 nmol liter−1 obtained for P. chrysosporium CPR (23), but it was 10-fold greater than the 7.5 nmol liter−1 recovered for native S. cerevisiae CPR (24). The kinetic properties of MgCPR using the one-electron reduction of oxidized cytochrome c with NADPH as a redox partner (Fig. 3) were similar to those observed previously with other eukaryotic CPR enzymes. In contrast to the one-electron reduction of cytochrome c, the reduction of cytochrome P450 monooxygenases by CPR is a two-electron process that involves two discrete one-electron transfers during the cytochrome P450 catalytic cycle (25). Previously, it has been shown that N-terminally truncated rat and human CPR enzymes could catalyze the reduction of cytochrome c but could not support cytochrome P450 catalysis (12, 26, 27). Therefore, it was important to establish that MgCPR also supported CYP51 catalysis. MgCPR acts as redox partner to C. albicans CYP51, but surprisingly, the redox pairing of MgCPR and MgCYP51 failed to demethylate both eburicol and lanosterol at 37°C. However, the MgCYP51-catalyzed demethylation of eburicol was observed by reducing the incubation temperature to 22°C (0.13 min−1), indicating that MgCYP51 catalysis was more temperature sensitive than C. albicans CYP51. These differences in temperature correlate to the biological growth of both organisms; C. albicans grows at 37°C in both the host and the laboratory, whereas the optimal growth temperature for M. graminicola is ∼25°C (mycelial growth). The inability of the MgCPR/MgCYP51 pairing to demethylate lanosterol suggests that MgCYP51 is an eburicol-dependent CYP51 enzyme, a finding supported by in silico experimentation. The modeling experiments suggested that lanosterol could fit into the active site, as supported by the type I binding spectra, but could not orientate the C-14-methyl group to facilitate catalysis, unlike for eburicol. This study is the first demonstration of selectivity and sterol specificity for a fungal CYP51, although obtusifoliol specificity (28) has been seen in plant and trypanosomal studies (29).
The MgCYP51 Km for eburicol was 3-fold higher than the previously calculated Ks value of 11 μM for eburicol binding to MgCYP51 by UV-visible spectroscopy (16) suggesting that the Km value is not solely dependent on the affinity of MgCYP51 for substrate. The MgCYP51 Km value for eburicol was comparable with that determined for human CYP51 of 30 μM with lanosterol (30) but was 4- to 6-fold larger than Km values previously determined for C. albicans and S. cerevisiae CYP51 enzymes (15, 31, 32), suggesting a degree of variability with regard to substrate affinity among CYP51 enzymes.
Previously, we have shown that MgCYP51 tightly bound agricultural azole fungicides (16) such as epoxiconazole (Kd, 17 nM), tebuconazole (Kd, 27 nM), and triadimenol (Kd, 30 nM), apart from prothioconazole, which bound weakly (Kd, 14 μM) in an atypical fashion (not through direct coordination with the heme iron). The IC50 determinations (Fig. 7) using the MgCPR/MgCYP51 pairing and eburicol as the substrate demonstrated that prothioconazole was a very poor inhibitor. Prothioconazole is one of the most economically important fungicides currently used and was introduced in 2004. This study showed no significant inhibition of CYP51 activity by prothioconazole at concentrations up to 145 μM, corresponding to the atypical weak binding previously observed (16). The desthio form of prothioconazole, however, was shown to be the most potent inhibitor of MgCYP51 (IC50 of ∼0.6 μM), confirming that this is the active derivative of prothioconazole. This is corroborated by the findings of Parker et al. (17) in studies with C. albicans that the antifungal effect observed was due to prothioconazole being readily metabolized to its desthio form rather than the fungicide itself.
This study has shown the existence of a novel fungal CYP51 enzyme that is not only substrate specific but also temperature-sensitive in the presence of its native CPR. The homologous reconstitution system which has been developed in this study enables future in vitro studies on the effects of individual MgCYP51 point mutations, observed in M. graminicola field isolates, on both the azole antifungal resistance and catalytic efficiency. It will also allow the potential effects of new DMIs to be evaluated. Furthermore, this study has confirmed that the desthio form of prothioconazole is the active fungicide.
ACKNOWLEDGMENTS
We are grateful to the Engineering and Physical Sciences Research Council National Mass Spectrometry Service Centre at Swansea University for assistance with sterol analysis.
This work was supported by the European Union European Regional Development Fund (ERDF) via the Wales European Funding Office of the Welsh Government through the BEACON funding initiative. W. David Nes was supported by National Science Foundation grant NSF-MCB-09020212.
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Information & Contributors
Information
Published In
Applied and Environmental Microbiology
Volume 81 • Number 10 • 15 May 2015
Pages: 3379 - 3386
Editor: J. L. Schottel
PubMed: 25746994
Copyright
© 2015 American Society for Microbiology.
History
Received: 7 December 2014
Accepted: 2 March 2015
Published online: 22 April 2015
Contributors
Editor
J. L. Schottel
Editor
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Citations
Citation
2015.
Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase. Appl Environ Microbiol
81:.
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