- Email: [email protected]
Cytochromes P450nor and P450foxy of the fungus Fusarium oxysporum
International Congress Series 1233 (2002) 89 – 97
Cytochromes P450nor and P450foxy of the fungus Fusarium oxysporum Hirofumi Shoun*, Naoki Takaya Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Abstract Fusarium oxysporum contains two self-sufficient P450 species, P450nor (CYP55) and P450foxy (CYP505), both of which can complete their catalyses without the aid of other proteinaceous components and receive electrons directly from NADH or NADPH. P450nor reduces nitric oxide to nitrous oxide and is essential for fungal denitrification. Its tertiary structure is fundamentally the same as those of other P450s, whereas characteristic features, the presence of a big space and a positive charge cluster in the heme-distal pocket, correspond to the unique catalytic mechanism of P450nor. We studied the reaction mechanism of P450nor by analyzing various mutant proteins and by carrying out X-ray crystallography to obtain the following conclusion: (1) The distal positive charge cluster plays a crucial role in attracting and binding NADH. (2) The specificity against NADH and NADPH is mainly determined by the BV-helix. (3) The anion hole near the heme creates an electrophilic environment for the electrons to be transferred from NADH to the heme. P450foxy catalyzes x-1fx-3 hydroxylation of fatty acids. Cloning and expression in the heterologous yeast cells of its gene showed that it is a fused protein of P450 and its reductase. It was also shown that not only the P450 domain, but also the reductase domain of P450foxy, exhibits high amino acid sequence identities to the respective domains of P450BM3 of Bacillus megaterium. As F. oxysporum and B. megaterium belong to eukaryote and prokaryote, respectively, occurrence of the fused proteins with high similarity across phyla evokes interest with respect to the evolution of the P450 superfamily. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; Cytochrome P450nor; Cytochrome P450foxy; Nitric oxide; Fatty acid; Fusarium oxysporum
* Corresponding author. Present address: Department of Biotechnology, Graduate School of Agricultural and Life Science, University of Tokyo, Yayoi Bunkyo-ku, 113-8657 Tokyo, Japan. E-mail address: [email protected] (H. Shoun).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 3 7 8 - 3
90
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
1. Introduction The imperfect fungus Fusarium oxysporum is widely distributed in soil and known as a plant pathogen. It is also known to be the first eukaryotic denitrifier that catalyzes the reduction of nitrate into gaseous nitrous oxide (N2O) [1]. During the last 10 years, many eukaryotic microorganisms, such as fungi and yeast, were found in our laboratory to exhibit denitrifying activities [2– 4] although occurrence of such activities were formerly believed to be restricted to only prokaryotes (bacteria) [5]. Denitrification together with nitrogen fixation and nitrification is important for comprising the global nitrogen cycle. Biochemical studies for denitrifying enzymes of the fungus F. oxysporum revealed reducing enzymes of nitrogen oxides such as nitrate reductase, nitrite reductase, and nitric oxide (NO) reductase [6– 8]. During the course of our biochemical studies on this fungus, one of the most outstanding progresses was the isolation of two unique cytochrome P450 species named P450nor and P450foxy (Fig. 1) [8 – 10]. They are quite distinct from other P450 oxidoreductases in that they require no electron-donating protein for catalytic activity. Furthermore, P450nor is a sole P450 that catabolizes NO reported so far and its catalytic activity has been shown to be essential for the denitrification by fungus. Here, we described a brief introduction and current research that is being carried out on these P450s.
2. Cytochrome P450nor P450nor was first purified from the fungus F. oxysporum MT-811 by using quasilipoxygenase activity as the index of purification [11]. It was shown after a latent period that the soluble P450 exhibited an intriguing feature, that it was induced by NO3 or NO2 under restricted aeration [12]. These results led us to the finding of the denitrification by the F. oxysporum strain MT-811 [2], and the soluble P450 was later characterized as NO reductase ( P450nor) [8]. Fungal denitrifiers widely occur across subdivisions of mycota as described below. At present, denitrifiers are found in fungi including: Gibberella fujikuroi, F. oxysporum, F. lini, F. decemcellulare, F. solani, Cylindrocarpon tonkinense, Trichoderma hamatum, Chaetomium sp., Talaromyces flavus, and Trichosporon cutaneum [2– 4]. These denitri-
Fig. 1. The P450 system of F. oxysporum.
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
91
Table 1 Distribution of fungal denitrifiers Subdivision
Class
Order
Family
Genus
Denitrification
Zygomycotina
Zygomycetes
Mucorales
Mucoraceae
Gymnoascales
Gymnoascales Monascaceae Trichomaceae
Pyrenomycetes
Sphaeriales
Melanosporaceae Sordariaceae Hypocreaceae
Teliomycetes Hymenomycetes
Ustilaginales Exobasidiales
Hypomycetaceae Sporidiaceae Firobasidaceae
Mucor Rhizopus Mannizzia Monascus Eupenicillium Aspergillus Penicillium Talaromyces Chaetomium Neurospora Calonectria Fusarium Gibberella Hypocrea Nectria Trichoderma Cylindrocarpon Hypomyces Rhodosporidium Filobasidium
++ ++ ++ ++ ++ + ++ ++ +
Ascomycotina
Bacidiomycotina
Plectomycetes
fiers are mainly distributed among the subdivision of ascomycotina although T. cutaneum was classified into basidiomycotina (Table 1). Among them, many F. oxysporum strains, G. fujikuroi, C. tonkinense, F. lini, and T. cutaneum produced in the P450 cells, and their cell-free extracts contained proteins that reacted with the antibodies against P450nor of F. oxysporum [11]. After P450nor of F. oxysporum was identified as NO reductase [8], its isozymes were purified from C. tonkinense [9] and T. cutaneum [10] and their corresponding genes were cloned [10,13,14]. All of the P450nor purified to date are soluble proteins of which molecular weights range from 42 to 46.4 kDa [8– 10]. The crystal structure of F. oxysporum P450nor has been determined and showed essentially a similar structure to other P450s [15]. Although P450nor is related to other P450s, its enzymic functions are surprising as it catalyzes the reduction of NO, whereas most P450s are known to catalyze the monooxygenation of lipophilic compounds [16]. The activity of P450nor could be reconstituted with the cellfree extracts of those fungi by using NAD(P)H as a sole electron donor [8 – 10]. Furthermore, P450nor by itself has no cofactor-mediating electrons to the heme moiety, indicating that direct electron transfer from NAD(P)H to the heme occurs in the catalytic cycle. This is a striking property because no direct electron transfer from NAD(P)H to the heme has been reported in any other hemoproteins. The catalytic mechanisms of P450nor have been extensively studied in F. oxysporum P450nor. The overall reaction can be divided into three partial reactions [13,14]. Fe3þ þ NO ! Fe3þ NO
Step 1
92
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
Fe3þ NO þ NADðPÞH ! I þ NADðPÞþ
Step 2
I þ NO þ Hþ ! Fe3þ þ N2 O þ H2 O:
Step 3
First, the ferric form (Fe3+) of P450nor binds the substrate NO to form a ferric-NO complex (Fe3+ – NO) (Step 1). Thereafter, Fe3+ –NO is reduced with NAD(P)H to form a specific intermediate (I) with a Soret absorption band at 444 nm (Step 2) and finally reacts with the second NO to form the product N2O (Step 3). The entire reaction proceeds at a high rate (above 1000 s1 at 10 jC) [8,17]. Recently, we analyzed the reaction in the (Step 2), the rate-limiting step in the NO reduction by P450nor, and clarified the redox mechanism in some aspects. The crystal structure of P450nor showed that the heme-distal pocket is in a highly hydrophilic environment and contained many water molecules as compared with those of other P450s [18]. A specific hydrogen bond network comprised of Water74, Ser286, Water33 and Asp393 is formed when NO binds to the resting heme
Fig. 2. The structure of the anion-binding site (Br2) in the heme-distal pocket.
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
93
(Step 1). Water74 is adjacent to the heme iron-bound NO and, thus, the network connects the active site to the bulk solvent and would act as a proton-delivery system when the Fe3+NO complex is reduced with NAD(P)H (Step 2). The crystal structure of P450nor also showed that a wide-open space is formed in the heme-distal pocket [18,19]. This heme-distal pocket is abnormally concentrated with positively charged Lys and Arg residues. We constructed a series of mutant proteins by site-directed mutagenesis to examine the roles of the positively charged cluster and showed that this unique cluster is responsible for the characteristic function of P450nor, possibly for direct interaction with NAD(P)H [19]. These results suggest that the positively charged cluster attracts the negatively charged NAD(P)H molecule by ionic interactions. Thus, direct electron transfer from NAD(P)H to the heme iron is supported by the interaction of NAD(P)H from the distal side of the heme [19]. With regard to this point, P450nor is unique because other P450 monooxygenases use the proximal side for transferring electrons from electron-mediating proteins. It has been shown that halogen anions induce a reverse type-1 spectral change in the heme moiety and inhibit catalytic activity of P450nor [19]. The binding sites of bromide anions (Br) in P450nor were revealed by X-ray crystallography (Fig. 2). Two Br were clearly observed in the heme-distal pocket. One of the binding sites is located near the heme and the polypeptide backbone around 289th Ala is bent upward at the binding site. This makes a binding hole for Br and thus is called the anion hole. This structure is likely to attract electrons to the heme moiety during the reducing step (Step 2), which is inhibited by halogen anions, and may be involved in transferring electrons from NADH.
3. Cytochrome P450foxy Fatty acid subterminal hydroxylase activity was detected in the cell-free extract of F. oxysporum and suggested to be a possible candidate for the physiological function of the purified P450nor [11]. It was shown after a long period that the fungus contains two distinct species of P450, which were recovered in the soluble and membrane fractions, respectively [20,21]. The soluble one has now been identified as P450nor described above, and another P450 recovered in the membrane fraction has now been characterized to be dependent with P450foxy that catalyzes fatty acid (x-1 to x-3) hydroxylation [21]. P450foxy completes its function without the aid of other proteinous components as well as P450nor. Recent studies on the cloning of the P450foxy-encoding gene (CYP505) [22] revealed that P450foxy was constituted from P450 and its reductase domains and the linker region between them like P450BM3, a bacterial isozyme catalyzing a similar reaction [23]. The predicted amino acid sequence of P450foxy was similar to that of P450BM3 and the sequence identity between them was as high as 37.3%, indicating that they belong to the same family in the P450 superfamily. The P450 domain showed the highest degree of identity to the P450 domain of P450BM3 (40.6%). It was also shown that they are more closely related to the CYP4, CYP52, and CYP86 families than other families [24], all of which catalyze fatty acid subterminal (or terminal) hydroxylations although the sequence identities to other P450s were below 24%. It is interesting that these fatty acid hydroxylase P450s form a cluster in the phylogenetic tree on the basis of the
94
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
catalytic function rather than the phylogenetic relationship of the organisms of their origins. As for the P450 reductase domain, P450foxy again showed the highest sequence identity (35.3%) to the P450 reductase domain of P450BM3 (Table 1) although the sequence identities to other P450 reductases were around 25%. These results demonstrated that P450foxy and P450BM3 might originate from the same ancient gene, which may be transferred to a recipient strain after the P450 and P450 reductase domains were fused. P450foxy also resembles P450BM3 in the Mr value and catalytic properties [21]. On the other hand, the difference between P450foxy and P450BM3 was in their intracellular localization. P450foxy was purified from the membrane fraction after solubilization [21] and not detected in the soluble fraction [22], whereas P450BM3 is a soluble protein. As the predicted amino acid sequence does not contain any hydrophobic stretch, the mechanism that anchors P450foxy to the membrane is to be elucidated. We have succeeded in producing recombinant P450foxy, which was catalytically and spectrally indistinguishable from the native protein, by using the heterologous host S. cerevisiae [22]. The recombinant protein mostly recovered in the soluble fraction of the yeast cells in contrast to the native protein that exclusively recovered in the membrane fraction of the fungal cells. No difference was detected by the SDS-PAGE between the Mr values of the native protein and the recombinant protein species recovered in the soluble or the membrane fraction. At present, we cannot rule out the possibility that a few amino acid residues might be removed from the N- or the C-terminus by some posttranslational proteolysis in the recombinant P450foxy. However, it is unlikely that a small portion of the polypeptide comprising of only a few amino acid residues can anchor the polypeptide to the membrane. On the other hand, native P450foxy would be targeted and bound to the membrane by some posttranslational (or co-translational) mechanism and functions in the fungal cells; however, it does not work in the yeast cells because it was exclusively recovered in the membrane fraction of the fungal cells. Modification of the protein with a hydrophobic moiety, such as a fatty acid or a prenyl group [24,25], would be possible. Such a modification is consistent with our results with native P450foxy that it is loosely bound to membrane and the purified protein was inert against Edman degradation performed by the automated peptide sequencer (Nakayama et al., unpublished data), suggesting that its Nterminus is blocked. In addition, the mechanism that targets and binds P450foxy to the membrane is still an area of interest, which needs to be investigated.
4. Future perspectives Recent progress in genome analysis revealed that P450nor and P450foxy are found in various types of fungi than previously supposed. At present, sequences homologous to the gene of F. oxysporum P450nor appear in the EST library of Neurospora crassa, Aspergillus flavus, and A. oryzae. These fungi, including F. oxysporum, belong to the subdivision ascomycotina, whereas T. cutaneum is a basidiomycetous fungus. These facts mean that P450nor along with denitrification are widely distributed among fungi and, thus, denitrification is the general, eukaryotic energy-acquisition system of fungi. This evokes an evolutional interest into when and how the eukaryote (fungi) acquired the anaerobic
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
95
Table 2 Similarities between P450foxy homologs Similarity (%) P450BM3 P450foxy P450BM3
P450 40.6 –
fum6p CPR 35.3
Entire 37.2
P450 42.5 P450 35.4
CPR 37.8 CPR 32.7
Entire 39.8 Entire 33.8
Similarity in the amino acid sequences of the P450 domains ( P450); the P450 reductase domains (CRP); whole proteins (entire).
respiration system. Furthermore, the results also evoke an interesting question concerning the molecular evolution of P450nor because P450nor is phylogenetically classified into the group of bacterial P450s [13] although its counterpart has not yet been found in bacteria in spite of the recent progress in genome analyses. Recently, some related sequences to P450foxy were found not only in B. megaterium, but also in B. subtilis, F. sporotrichioides, and Giberrela moniliformis (Table 2). It is surprising that P450foxy is distributed in the restricted microorganisms including the Bacillus species and ascomycetous fungi. Recent results showed that the fum6 gene of G. moniliformis encodes the P450foxy homolog and is located in the gene cluster involved in mycotoxin biosynthesis. The mycotoxin (fumonisin) is comprised of a set of compounds structurally similar to fatty acids on which carbon atoms are hydroxylated or methoxylated [26]. It is easily supposed that this P450foxy homolog may introduce oxygen atoms to the substrate fatty acid analogues by monooxygenation reaction. F. oxysporum and G. moniliformis are related to each other, suggesting that F. oxysporum P450foxy might function in a similar manner. Future investigations on fumonisin production by F. oxysporum are therefore important. It is interesting in the evolutional aspect that both F. oxysporum and B. megaterium share P450foxy ( P450BM3) as they are phylogenetically distant and belong to eukaryote and prokaryote, respectively. There is no report that the bacterium secretes a fumonisin-like mycotoxin. Now that we have succeeded in producing recombinant preparation of F. oxysporum P450foxy, future biochemical studies on this interesting enzyme will hopefully shed light on the physiological functions of P450foxy.
Acknowledgements This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and the Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan.
References [1] W.G. Zumft, Cell biology and molecular basis of denitrification, Microbiol. Mol. Biol. Rev. 61 (1997) 533 – 616.
96
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
[2] H. Shoun, T. Tanimoto, Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction, J. Biol. Chem. 266 (1991) 11078 – 11082. [3] S. Tsuruta, N. Takaya, L. Zhang, H. Shoun, K. Kimura, M. Hamamoto, T. Nakase, Denitrification by yeasts and occurrence of cytochrome P450nor in Trichosporon cutaneum, FEMS Microbiol. Lett. 168 (1998) 105 – 110. [4] H. Shoun, D.H. Kim, H. Uchiyama, J. Sugiyama, Denitrification by fungi, FEMS Microbiol. Lett. 94 (1992) 277 – 282. [5] B.A. Averill, Dissimilatory nitrite and nitric oxide reductases, Chem. Rev. 96 (1996) 2951 – 2964. [6] M. Kobayashi, H. Shoun, The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus Fusarium oxysporum, J. Biol. Chem. 270 (1995) 4146 – 4151. [7] M. Kobayashi, Y. Matsuo, A. Takimoto, S. Suzuki, F. Maruo, H. Shoun, Denitrification, a novel type of respiratory metabolism in fungal mitochondrion, J. Biol. Chem. 271 (1996) 16263 – 16267. [8] K. Nakahara, T. Tanimoto, K. Hatano, K. Usuda, H. Shoun, Cytochrome P-450 55A1 ( P-450dNIR) acts as nitric oxide reductase employing NADH as the direct electron donor, J. Biol. Chem. 268 (1993) 8350 – 8355. [9] K. Usuda, N. Toritsuka, Y. Matsuo, D.H. Kim, H. Shoun, Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor, Appl. Environ. Microbiol. 61 (1995) 883 – 889. [10] L. Zhang, N. Takaya, T. Kitazume, T. Kondo, H. Shoun, Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum, Eur. J. Biochem. 268 (2001) 3198 – 3204. [11] H. Shoun, Y. Sudo, Y. Seto, T. Beppu, Purification and properties of a cytochrome P-450 of a fungus, Fusarium oxysporum, J. Biochem. (Tokyo) 94 (1983) 1219 – 1229. [12] H. Shoun, W. Suyama, T. Yasui, Soluble, nitrate/nitrite-inducible cytochrome P-450 of the fungus, Fusarium oxysporum, FEBS Lett. 244 (1989) 11 – 14. [13] H. Kizawa, D. Tomura, M. Oda, A. Fukamizu, T. Hoshino, O. Gotoh, T. Yasui, H. Shoun, Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum, J. Biol. Chem. 266 (1991) 10632 – 10637. [14] T. Kudo, D. Tomura, D.L. Liu, X.Q. Dai, H. Shoun, Two isozymes of P450nor of Cylindrocarpon tonkinense: molecular cloning of the cDNAs and genes, expressions in the yeast, and the putative NAD(P)Hbinding site, Biochimie 78 (1996) 792 – 799. [15] S.Y. Park, H. Shimizu, S. Adachi, A. Nakagawa, I. Tanaka, K. Nakahara, H. Shoun, H. Nakamura, T. Iizuka, Y. Shiro, Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum, Nat. Struct. Biol. 4 (1997) 827 – 832. [16] T. Omura, Forty years of cytochrome P450, Biochem. Biophys. Res. Commun. 233 (1999) 690 – 698. [17] Y. Shiro, M. Fujii, T. Iizuka, S. Adachi, K. Tsukamoto, K. Nakahara, H. Shoun, Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide, J. Biol. Chem. 270 (1995) 1617 – 1623. [18] H. Shimizu, E. Obayashi, Y. Gomi, H. Arakawa, S.Y. Park, H. Nakamura, S. Adachi, H. Shoun, Y. Shiro, Proton delivery in NO reduction by fungal nitric-oxide reductase: cryogenic crystallography, spectroscopy, and kinetics of ferric-NO complexes of wild-type and mutant enzymes, J. Biol. Chem. 275 (2000) 4816 – 4826. [19] T. Kudo, N. Takaya, S.Y. Park, Y. Shiro, H. Shoun, A positively charged cluster formed in the hemedistal pocket of cytochrome P450nor is essential for interaction with NADH, J. Biol. Chem. 276 (2001) 5020 – 5026. [20] H. Shoun, Y. Sudo, T. Beppu, Subterminal hydroxylation of fatty acids by a cytochrome P-450-dependent enzyme system from a fungus, Fusarium oxysporum, J. Biochem. (Tokyo) 97 (1985) 755 – 763. [21] N. Nakayama, A. Takemae, H. Shoun, Fatty acid hydroxylase of the fungus Fusarium oxysporum is possibly a fused protein of cytochrome P-450 and its reductase (1996), J. Biochem. (Tokyo) 119 (1996) 435 – 440. [22] T. Kitazume, N. Takaya, N. Nakayama, H. Shoun, Cytochrome P450foxy, a catalytically self-sufficient fatty acid hydroxylase of the fungus Fusarium oxysporum, J. Biol. Chem. 275 (2000) 39734 – 39740. [23] L.O. Narhi, A.J. Fulco, Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium, J. Biol. Chem. 261 (1986) 7160 – 7169.
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
97
[24] R.J.A. Grand, Acylation of viral and eukaryotic proteins, Biochem. J. 258 (1989) 625 – 638. [25] J.A. Glomset, M.H. Gelb, C.C. Farnsworth, Prenyl proteins in eukaryotic cells: a new type of membrane anchor, Trends Biol. Sci. 15 (1990) 139 – 142. [26] R.H. Proctor, A.E. Desjardins, R.D. Plattner, T.M. Hohn, Polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A, Fungal Genet. Biol. 27 (1999) 100 – 112.
Cytochromes P450nor and P450foxy of the fungus Fusarium oxysporum Hirofumi Shoun*, Naoki Takaya Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Abstract Fusarium oxysporum contains two self-sufficient P450 species, P450nor (CYP55) and P450foxy (CYP505), both of which can complete their catalyses without the aid of other proteinaceous components and receive electrons directly from NADH or NADPH. P450nor reduces nitric oxide to nitrous oxide and is essential for fungal denitrification. Its tertiary structure is fundamentally the same as those of other P450s, whereas characteristic features, the presence of a big space and a positive charge cluster in the heme-distal pocket, correspond to the unique catalytic mechanism of P450nor. We studied the reaction mechanism of P450nor by analyzing various mutant proteins and by carrying out X-ray crystallography to obtain the following conclusion: (1) The distal positive charge cluster plays a crucial role in attracting and binding NADH. (2) The specificity against NADH and NADPH is mainly determined by the BV-helix. (3) The anion hole near the heme creates an electrophilic environment for the electrons to be transferred from NADH to the heme. P450foxy catalyzes x-1fx-3 hydroxylation of fatty acids. Cloning and expression in the heterologous yeast cells of its gene showed that it is a fused protein of P450 and its reductase. It was also shown that not only the P450 domain, but also the reductase domain of P450foxy, exhibits high amino acid sequence identities to the respective domains of P450BM3 of Bacillus megaterium. As F. oxysporum and B. megaterium belong to eukaryote and prokaryote, respectively, occurrence of the fused proteins with high similarity across phyla evokes interest with respect to the evolution of the P450 superfamily. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; Cytochrome P450nor; Cytochrome P450foxy; Nitric oxide; Fatty acid; Fusarium oxysporum
* Corresponding author. Present address: Department of Biotechnology, Graduate School of Agricultural and Life Science, University of Tokyo, Yayoi Bunkyo-ku, 113-8657 Tokyo, Japan. E-mail address: [email protected] (H. Shoun).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 3 7 8 - 3
90
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
1. Introduction The imperfect fungus Fusarium oxysporum is widely distributed in soil and known as a plant pathogen. It is also known to be the first eukaryotic denitrifier that catalyzes the reduction of nitrate into gaseous nitrous oxide (N2O) [1]. During the last 10 years, many eukaryotic microorganisms, such as fungi and yeast, were found in our laboratory to exhibit denitrifying activities [2– 4] although occurrence of such activities were formerly believed to be restricted to only prokaryotes (bacteria) [5]. Denitrification together with nitrogen fixation and nitrification is important for comprising the global nitrogen cycle. Biochemical studies for denitrifying enzymes of the fungus F. oxysporum revealed reducing enzymes of nitrogen oxides such as nitrate reductase, nitrite reductase, and nitric oxide (NO) reductase [6– 8]. During the course of our biochemical studies on this fungus, one of the most outstanding progresses was the isolation of two unique cytochrome P450 species named P450nor and P450foxy (Fig. 1) [8 – 10]. They are quite distinct from other P450 oxidoreductases in that they require no electron-donating protein for catalytic activity. Furthermore, P450nor is a sole P450 that catabolizes NO reported so far and its catalytic activity has been shown to be essential for the denitrification by fungus. Here, we described a brief introduction and current research that is being carried out on these P450s.
2. Cytochrome P450nor P450nor was first purified from the fungus F. oxysporum MT-811 by using quasilipoxygenase activity as the index of purification [11]. It was shown after a latent period that the soluble P450 exhibited an intriguing feature, that it was induced by NO3 or NO2 under restricted aeration [12]. These results led us to the finding of the denitrification by the F. oxysporum strain MT-811 [2], and the soluble P450 was later characterized as NO reductase ( P450nor) [8]. Fungal denitrifiers widely occur across subdivisions of mycota as described below. At present, denitrifiers are found in fungi including: Gibberella fujikuroi, F. oxysporum, F. lini, F. decemcellulare, F. solani, Cylindrocarpon tonkinense, Trichoderma hamatum, Chaetomium sp., Talaromyces flavus, and Trichosporon cutaneum [2– 4]. These denitri-
Fig. 1. The P450 system of F. oxysporum.
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
91
Table 1 Distribution of fungal denitrifiers Subdivision
Class
Order
Family
Genus
Denitrification
Zygomycotina
Zygomycetes
Mucorales
Mucoraceae
Gymnoascales
Gymnoascales Monascaceae Trichomaceae
Pyrenomycetes
Sphaeriales
Melanosporaceae Sordariaceae Hypocreaceae
Teliomycetes Hymenomycetes
Ustilaginales Exobasidiales
Hypomycetaceae Sporidiaceae Firobasidaceae
Mucor Rhizopus Mannizzia Monascus Eupenicillium Aspergillus Penicillium Talaromyces Chaetomium Neurospora Calonectria Fusarium Gibberella Hypocrea Nectria Trichoderma Cylindrocarpon Hypomyces Rhodosporidium Filobasidium
++ ++ ++ ++ ++ + ++ ++ +
Ascomycotina
Bacidiomycotina
Plectomycetes
fiers are mainly distributed among the subdivision of ascomycotina although T. cutaneum was classified into basidiomycotina (Table 1). Among them, many F. oxysporum strains, G. fujikuroi, C. tonkinense, F. lini, and T. cutaneum produced in the P450 cells, and their cell-free extracts contained proteins that reacted with the antibodies against P450nor of F. oxysporum [11]. After P450nor of F. oxysporum was identified as NO reductase [8], its isozymes were purified from C. tonkinense [9] and T. cutaneum [10] and their corresponding genes were cloned [10,13,14]. All of the P450nor purified to date are soluble proteins of which molecular weights range from 42 to 46.4 kDa [8– 10]. The crystal structure of F. oxysporum P450nor has been determined and showed essentially a similar structure to other P450s [15]. Although P450nor is related to other P450s, its enzymic functions are surprising as it catalyzes the reduction of NO, whereas most P450s are known to catalyze the monooxygenation of lipophilic compounds [16]. The activity of P450nor could be reconstituted with the cellfree extracts of those fungi by using NAD(P)H as a sole electron donor [8 – 10]. Furthermore, P450nor by itself has no cofactor-mediating electrons to the heme moiety, indicating that direct electron transfer from NAD(P)H to the heme occurs in the catalytic cycle. This is a striking property because no direct electron transfer from NAD(P)H to the heme has been reported in any other hemoproteins. The catalytic mechanisms of P450nor have been extensively studied in F. oxysporum P450nor. The overall reaction can be divided into three partial reactions [13,14]. Fe3þ þ NO ! Fe3þ NO
Step 1
92
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
Fe3þ NO þ NADðPÞH ! I þ NADðPÞþ
Step 2
I þ NO þ Hþ ! Fe3þ þ N2 O þ H2 O:
Step 3
First, the ferric form (Fe3+) of P450nor binds the substrate NO to form a ferric-NO complex (Fe3+ – NO) (Step 1). Thereafter, Fe3+ –NO is reduced with NAD(P)H to form a specific intermediate (I) with a Soret absorption band at 444 nm (Step 2) and finally reacts with the second NO to form the product N2O (Step 3). The entire reaction proceeds at a high rate (above 1000 s1 at 10 jC) [8,17]. Recently, we analyzed the reaction in the (Step 2), the rate-limiting step in the NO reduction by P450nor, and clarified the redox mechanism in some aspects. The crystal structure of P450nor showed that the heme-distal pocket is in a highly hydrophilic environment and contained many water molecules as compared with those of other P450s [18]. A specific hydrogen bond network comprised of Water74, Ser286, Water33 and Asp393 is formed when NO binds to the resting heme
Fig. 2. The structure of the anion-binding site (Br2) in the heme-distal pocket.
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
93
(Step 1). Water74 is adjacent to the heme iron-bound NO and, thus, the network connects the active site to the bulk solvent and would act as a proton-delivery system when the Fe3+NO complex is reduced with NAD(P)H (Step 2). The crystal structure of P450nor also showed that a wide-open space is formed in the heme-distal pocket [18,19]. This heme-distal pocket is abnormally concentrated with positively charged Lys and Arg residues. We constructed a series of mutant proteins by site-directed mutagenesis to examine the roles of the positively charged cluster and showed that this unique cluster is responsible for the characteristic function of P450nor, possibly for direct interaction with NAD(P)H [19]. These results suggest that the positively charged cluster attracts the negatively charged NAD(P)H molecule by ionic interactions. Thus, direct electron transfer from NAD(P)H to the heme iron is supported by the interaction of NAD(P)H from the distal side of the heme [19]. With regard to this point, P450nor is unique because other P450 monooxygenases use the proximal side for transferring electrons from electron-mediating proteins. It has been shown that halogen anions induce a reverse type-1 spectral change in the heme moiety and inhibit catalytic activity of P450nor [19]. The binding sites of bromide anions (Br) in P450nor were revealed by X-ray crystallography (Fig. 2). Two Br were clearly observed in the heme-distal pocket. One of the binding sites is located near the heme and the polypeptide backbone around 289th Ala is bent upward at the binding site. This makes a binding hole for Br and thus is called the anion hole. This structure is likely to attract electrons to the heme moiety during the reducing step (Step 2), which is inhibited by halogen anions, and may be involved in transferring electrons from NADH.
3. Cytochrome P450foxy Fatty acid subterminal hydroxylase activity was detected in the cell-free extract of F. oxysporum and suggested to be a possible candidate for the physiological function of the purified P450nor [11]. It was shown after a long period that the fungus contains two distinct species of P450, which were recovered in the soluble and membrane fractions, respectively [20,21]. The soluble one has now been identified as P450nor described above, and another P450 recovered in the membrane fraction has now been characterized to be dependent with P450foxy that catalyzes fatty acid (x-1 to x-3) hydroxylation [21]. P450foxy completes its function without the aid of other proteinous components as well as P450nor. Recent studies on the cloning of the P450foxy-encoding gene (CYP505) [22] revealed that P450foxy was constituted from P450 and its reductase domains and the linker region between them like P450BM3, a bacterial isozyme catalyzing a similar reaction [23]. The predicted amino acid sequence of P450foxy was similar to that of P450BM3 and the sequence identity between them was as high as 37.3%, indicating that they belong to the same family in the P450 superfamily. The P450 domain showed the highest degree of identity to the P450 domain of P450BM3 (40.6%). It was also shown that they are more closely related to the CYP4, CYP52, and CYP86 families than other families [24], all of which catalyze fatty acid subterminal (or terminal) hydroxylations although the sequence identities to other P450s were below 24%. It is interesting that these fatty acid hydroxylase P450s form a cluster in the phylogenetic tree on the basis of the
94
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
catalytic function rather than the phylogenetic relationship of the organisms of their origins. As for the P450 reductase domain, P450foxy again showed the highest sequence identity (35.3%) to the P450 reductase domain of P450BM3 (Table 1) although the sequence identities to other P450 reductases were around 25%. These results demonstrated that P450foxy and P450BM3 might originate from the same ancient gene, which may be transferred to a recipient strain after the P450 and P450 reductase domains were fused. P450foxy also resembles P450BM3 in the Mr value and catalytic properties [21]. On the other hand, the difference between P450foxy and P450BM3 was in their intracellular localization. P450foxy was purified from the membrane fraction after solubilization [21] and not detected in the soluble fraction [22], whereas P450BM3 is a soluble protein. As the predicted amino acid sequence does not contain any hydrophobic stretch, the mechanism that anchors P450foxy to the membrane is to be elucidated. We have succeeded in producing recombinant P450foxy, which was catalytically and spectrally indistinguishable from the native protein, by using the heterologous host S. cerevisiae [22]. The recombinant protein mostly recovered in the soluble fraction of the yeast cells in contrast to the native protein that exclusively recovered in the membrane fraction of the fungal cells. No difference was detected by the SDS-PAGE between the Mr values of the native protein and the recombinant protein species recovered in the soluble or the membrane fraction. At present, we cannot rule out the possibility that a few amino acid residues might be removed from the N- or the C-terminus by some posttranslational proteolysis in the recombinant P450foxy. However, it is unlikely that a small portion of the polypeptide comprising of only a few amino acid residues can anchor the polypeptide to the membrane. On the other hand, native P450foxy would be targeted and bound to the membrane by some posttranslational (or co-translational) mechanism and functions in the fungal cells; however, it does not work in the yeast cells because it was exclusively recovered in the membrane fraction of the fungal cells. Modification of the protein with a hydrophobic moiety, such as a fatty acid or a prenyl group [24,25], would be possible. Such a modification is consistent with our results with native P450foxy that it is loosely bound to membrane and the purified protein was inert against Edman degradation performed by the automated peptide sequencer (Nakayama et al., unpublished data), suggesting that its Nterminus is blocked. In addition, the mechanism that targets and binds P450foxy to the membrane is still an area of interest, which needs to be investigated.
4. Future perspectives Recent progress in genome analysis revealed that P450nor and P450foxy are found in various types of fungi than previously supposed. At present, sequences homologous to the gene of F. oxysporum P450nor appear in the EST library of Neurospora crassa, Aspergillus flavus, and A. oryzae. These fungi, including F. oxysporum, belong to the subdivision ascomycotina, whereas T. cutaneum is a basidiomycetous fungus. These facts mean that P450nor along with denitrification are widely distributed among fungi and, thus, denitrification is the general, eukaryotic energy-acquisition system of fungi. This evokes an evolutional interest into when and how the eukaryote (fungi) acquired the anaerobic
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
95
Table 2 Similarities between P450foxy homologs Similarity (%) P450BM3 P450foxy P450BM3
P450 40.6 –
fum6p CPR 35.3
Entire 37.2
P450 42.5 P450 35.4
CPR 37.8 CPR 32.7
Entire 39.8 Entire 33.8
Similarity in the amino acid sequences of the P450 domains ( P450); the P450 reductase domains (CRP); whole proteins (entire).
respiration system. Furthermore, the results also evoke an interesting question concerning the molecular evolution of P450nor because P450nor is phylogenetically classified into the group of bacterial P450s [13] although its counterpart has not yet been found in bacteria in spite of the recent progress in genome analyses. Recently, some related sequences to P450foxy were found not only in B. megaterium, but also in B. subtilis, F. sporotrichioides, and Giberrela moniliformis (Table 2). It is surprising that P450foxy is distributed in the restricted microorganisms including the Bacillus species and ascomycetous fungi. Recent results showed that the fum6 gene of G. moniliformis encodes the P450foxy homolog and is located in the gene cluster involved in mycotoxin biosynthesis. The mycotoxin (fumonisin) is comprised of a set of compounds structurally similar to fatty acids on which carbon atoms are hydroxylated or methoxylated [26]. It is easily supposed that this P450foxy homolog may introduce oxygen atoms to the substrate fatty acid analogues by monooxygenation reaction. F. oxysporum and G. moniliformis are related to each other, suggesting that F. oxysporum P450foxy might function in a similar manner. Future investigations on fumonisin production by F. oxysporum are therefore important. It is interesting in the evolutional aspect that both F. oxysporum and B. megaterium share P450foxy ( P450BM3) as they are phylogenetically distant and belong to eukaryote and prokaryote, respectively. There is no report that the bacterium secretes a fumonisin-like mycotoxin. Now that we have succeeded in producing recombinant preparation of F. oxysporum P450foxy, future biochemical studies on this interesting enzyme will hopefully shed light on the physiological functions of P450foxy.
Acknowledgements This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and the Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan.
References [1] W.G. Zumft, Cell biology and molecular basis of denitrification, Microbiol. Mol. Biol. Rev. 61 (1997) 533 – 616.
96
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
[2] H. Shoun, T. Tanimoto, Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction, J. Biol. Chem. 266 (1991) 11078 – 11082. [3] S. Tsuruta, N. Takaya, L. Zhang, H. Shoun, K. Kimura, M. Hamamoto, T. Nakase, Denitrification by yeasts and occurrence of cytochrome P450nor in Trichosporon cutaneum, FEMS Microbiol. Lett. 168 (1998) 105 – 110. [4] H. Shoun, D.H. Kim, H. Uchiyama, J. Sugiyama, Denitrification by fungi, FEMS Microbiol. Lett. 94 (1992) 277 – 282. [5] B.A. Averill, Dissimilatory nitrite and nitric oxide reductases, Chem. Rev. 96 (1996) 2951 – 2964. [6] M. Kobayashi, H. Shoun, The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus Fusarium oxysporum, J. Biol. Chem. 270 (1995) 4146 – 4151. [7] M. Kobayashi, Y. Matsuo, A. Takimoto, S. Suzuki, F. Maruo, H. Shoun, Denitrification, a novel type of respiratory metabolism in fungal mitochondrion, J. Biol. Chem. 271 (1996) 16263 – 16267. [8] K. Nakahara, T. Tanimoto, K. Hatano, K. Usuda, H. Shoun, Cytochrome P-450 55A1 ( P-450dNIR) acts as nitric oxide reductase employing NADH as the direct electron donor, J. Biol. Chem. 268 (1993) 8350 – 8355. [9] K. Usuda, N. Toritsuka, Y. Matsuo, D.H. Kim, H. Shoun, Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor, Appl. Environ. Microbiol. 61 (1995) 883 – 889. [10] L. Zhang, N. Takaya, T. Kitazume, T. Kondo, H. Shoun, Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum, Eur. J. Biochem. 268 (2001) 3198 – 3204. [11] H. Shoun, Y. Sudo, Y. Seto, T. Beppu, Purification and properties of a cytochrome P-450 of a fungus, Fusarium oxysporum, J. Biochem. (Tokyo) 94 (1983) 1219 – 1229. [12] H. Shoun, W. Suyama, T. Yasui, Soluble, nitrate/nitrite-inducible cytochrome P-450 of the fungus, Fusarium oxysporum, FEBS Lett. 244 (1989) 11 – 14. [13] H. Kizawa, D. Tomura, M. Oda, A. Fukamizu, T. Hoshino, O. Gotoh, T. Yasui, H. Shoun, Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum, J. Biol. Chem. 266 (1991) 10632 – 10637. [14] T. Kudo, D. Tomura, D.L. Liu, X.Q. Dai, H. Shoun, Two isozymes of P450nor of Cylindrocarpon tonkinense: molecular cloning of the cDNAs and genes, expressions in the yeast, and the putative NAD(P)Hbinding site, Biochimie 78 (1996) 792 – 799. [15] S.Y. Park, H. Shimizu, S. Adachi, A. Nakagawa, I. Tanaka, K. Nakahara, H. Shoun, H. Nakamura, T. Iizuka, Y. Shiro, Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum, Nat. Struct. Biol. 4 (1997) 827 – 832. [16] T. Omura, Forty years of cytochrome P450, Biochem. Biophys. Res. Commun. 233 (1999) 690 – 698. [17] Y. Shiro, M. Fujii, T. Iizuka, S. Adachi, K. Tsukamoto, K. Nakahara, H. Shoun, Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide, J. Biol. Chem. 270 (1995) 1617 – 1623. [18] H. Shimizu, E. Obayashi, Y. Gomi, H. Arakawa, S.Y. Park, H. Nakamura, S. Adachi, H. Shoun, Y. Shiro, Proton delivery in NO reduction by fungal nitric-oxide reductase: cryogenic crystallography, spectroscopy, and kinetics of ferric-NO complexes of wild-type and mutant enzymes, J. Biol. Chem. 275 (2000) 4816 – 4826. [19] T. Kudo, N. Takaya, S.Y. Park, Y. Shiro, H. Shoun, A positively charged cluster formed in the hemedistal pocket of cytochrome P450nor is essential for interaction with NADH, J. Biol. Chem. 276 (2001) 5020 – 5026. [20] H. Shoun, Y. Sudo, T. Beppu, Subterminal hydroxylation of fatty acids by a cytochrome P-450-dependent enzyme system from a fungus, Fusarium oxysporum, J. Biochem. (Tokyo) 97 (1985) 755 – 763. [21] N. Nakayama, A. Takemae, H. Shoun, Fatty acid hydroxylase of the fungus Fusarium oxysporum is possibly a fused protein of cytochrome P-450 and its reductase (1996), J. Biochem. (Tokyo) 119 (1996) 435 – 440. [22] T. Kitazume, N. Takaya, N. Nakayama, H. Shoun, Cytochrome P450foxy, a catalytically self-sufficient fatty acid hydroxylase of the fungus Fusarium oxysporum, J. Biol. Chem. 275 (2000) 39734 – 39740. [23] L.O. Narhi, A.J. Fulco, Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium, J. Biol. Chem. 261 (1986) 7160 – 7169.
H. Shoun, N. Takaya / International Congress Series 1233 (2002) 89–97
97
[24] R.J.A. Grand, Acylation of viral and eukaryotic proteins, Biochem. J. 258 (1989) 625 – 638. [25] J.A. Glomset, M.H. Gelb, C.C. Farnsworth, Prenyl proteins in eukaryotic cells: a new type of membrane anchor, Trends Biol. Sci. 15 (1990) 139 – 142. [26] R.H. Proctor, A.E. Desjardins, R.D. Plattner, T.M. Hohn, Polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A, Fungal Genet. Biol. 27 (1999) 100 – 112.