112NISR RESEARCH GRANT
2003 Research Grant

Basic Studies on Fungal Unique P450s for Industrial Applications
Hirofumi SHOUN
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo
We isolated two unique cytochromes P450, P450nor andP450foxy, from the fungus Fusarium oxysporum. P450nor
is involved in fungal denitrification and functions as nitricoxide (NO) reductase (Nor). P450foxy catalyzes subterminal (w -1w -3) hydroxylation of fatty acids. It is unique inthat it is a fused protein consisting of P450 and its reductase domains. P450 is an attractive material not only forbasic research but also in the aspect of industrial application of biological reactions. P450 usually catalyzes amonooxygenase reaction, as below.
SO2NAD(P)HH → SOH2ONAD(P)
The reaction is usually supported by other protein(s) thattransport two electrons of NAD(P)H to P450 (redox partner), since P450 cannot receive the electrons directly fromNAD(P)H. Many of eukaryotic P450s are bound to endoplasmic reticulum membrane, and their reactions are supported by their redox partner, NADPH-cytochrome P450
reductase (P450 reductase). P450 reductase is comprisedof, from its N-terminus, FMN-, FAD-, and NADPH-binding domains (Fig. 1). Electrons are transferred in the orderof: NADPH → FAD → FMN → P450. A single eukaryoticorganism usually contains many P450 species while it contains only one P450 reductase species (two species arefound in plants). Therefore, a single P450 reductase supports many P450 species, several tenth and in some cases,
several hundreds of P450 species. P450 and P450 reductase
is usually separate proteins, while in rare case, they arefused as a single polypeptide chain. P450foxy is the first instance of such a fused protein among eukaryotes (Fig. 1).

Fig. 1.

On the other hand, P450nor is not a monooxygenase butcatalyzes the following reaction as Nor.
2NONAD(P)HH → N2OH2ONAD(P)
Surprisingly, P450nor does not need a redox partner, andthus is the only P450 species that can receive electrons directly from NAD(P)H. P450-catalyzed reactions are extremely diversified, and therefore, are very attractive in theaspect of industrial application. However, P450 reactions
are generally difficult to be reconstituted because of theircomplex redox system, and their catalytic turnover is usually low. Such properties of P450 enzyme system make ahigh barrier for industrial application. On the other hand,
fused P450 proteins such as P450foxy and P450BM3 realize a high catalytic turnover possibly because the intramolecular electron transfer is faster than the intermolecular electron transfer. Further, P450nor exhibits exceptionally highcatalytic turnover as P450 (in the order of 100,000 min1).
Therefore, elucidation of the reaction mechanisms ofP450nor and P450foxy that exhibit exceptionally highspeed catalyses, are important also for industrial application.
Determination of crystal structure of P450nor in complex with an NAD analogue (NAAD)
P450nor is the only hemeprotein (or even the only metalloprotein) that can receive electrons directly from the physiological electron donor NADH or NADPH. Such an electron-transferring mechanism is unprecedented, because twoStructures of several types of P450 and its redox partner (P450 reductase).

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electron-transfer between NAD and its redox couple is conducted by transporting two electrons as a hydride ion (H)and thus two electrons are released simultaneously whereasa hemeprotein that contains only one electron-redox center(heme) can never receive H directly. Crystal structure ofP450nor in several forms, that is, resting ferric (Fe3), CObound (Fe2–CO), and NO-bound (Fe3–NO) forms, havebeen so far determined. Here we succeeded in obtaining acrystal of P450nor in complex with NAAD and determining its structure, as shown in Fig. 2, which provided conclusive evidence for the direct H transfer from NADH toP450nor.
(1) Whole structure: A global conformational changeof P450nor protein was observed upon binding of NAAD,
which was accompanied by various intriguing local movements. Entrance of the heme pocket was closed, while the Ihelix near heme moved a little away from heme. Thesechanges are typical of the “induced fit”.
(2) NADH-binding site: The heme-distal pocket iscomprised of three sides, as shown in Fig. 3. One is extended beneath the B-helix, in which many hydrophilicamino acid residues are concentrated (hydrophilic side). On
the other hand, the area beneath the F,G-loop is hydrophobic. These two sides form the NADH-access channel. The
third side forms a proton channel.
Arg174 is the only charged amino acid residue on the hydrophobic side. By contrast, the hydrophilic side contains
many charged groups; Glu71, Arg64, Asp88, Lys291,Fig. 2. Crystal structure of P450nor.
Two structures of P450nor in complex with NO (green) and with NAAD
(nicotinic acid adenine dinucleotide) (red) superimposed centering heme
(yellow). The B-helix and F,G-loop moved by 3.5 and 4.0 Å, respectively,
upon binding NAAD so that the entrance of the heme-distal pocket isclosed. NAAD is shown by red, bold line.

Fig. 3. NADH channel (bold, green circle) and proton channel (small, red circle).
A, A view from the heme-distal side perpendicular to the heme plate. B, Parallel to heme (bottom). Red, small spheres represent water. Side chains of Arg64
and Arg174, Glu71 and Asp88, Ser286 and Thr243, are depicted in blue, red, and green. NAAD is depicted by bold, green line.

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Lys62, and Arg292. As expected, Arg174 and Arg64 bindthe pyrophosphate moiety of NAAD between them. Thr243
in the I-helix binds the side chain of nicotinic acid ring andthus is crucial for stereo-selective hydrogen transfer from
NADH. Also as expected, Ser73 and Ser75 are located nearthe 2’-hydroxyl group of adenine ring, and thus play thecrucial role in determining the electron donor specificity,
NADH or/and NADPH. Arg292 binds one of the propionate side chains of heme. Arg292 accompanies the mass
movement of the charge cluster in the hydrophilic side thatis caused upon binding of NAAD. The marked movementof Arg292 is accompanied by upward shift of the propionate side chain of heme, which play crucial role in fixing
nicotinic acid ring in a stereo-selective manner.
(3) Active site: C4 carbon atom on nicotinic acid ringis located in the close vicinity of the hemebound NO andfaces its proR side to heme (NO). This stereo-selective
binding is mainly supported by Thr243 and the propionate
side chain, as noted above. This structure of NAAD-binding is consistent with our previous results on such as kinetic
isotope effect of hydrogen atoms on C4 of NADH, strongly
supporting the direct H transfer from NADH to the Fe3–NO complex. In parallel with the NADH channel is formeda proton channel that is comprised of Asp393, Ser286, andmany water molecules. The simultaneous formation of theproton channel upon binding NAAD suggests occurrenceof a proton transfer subsequent to the H transfer to form
the intermediate (equivalent to Fe3-hydroxylamine complex).
(4) Salt bridge network Glu71-Arg64-Asp88: In theNAAD-free structures of P450nor three charged groups,Glu71, Arg64, and Asp88 form a salt bridge network. Uponbinding of NAAD, Glu71 and Arg64 accompany the mass
movement of the charge cluster beneath the B-helix,
whereas Asp88 does not move so much. This means thatthe salt bridge between Arg64 and Asp88 is cut off upon
the NAAD binding. It would appear that formation of thesalt bridge network stabilizes the protein structure andbreakage of the network is energetically unfavorable. This
results in the low affinity of P450nor against NADH/NAD
by increasing the rate of release of NAD (koff), but in realizing the high catalytic turnover. That is, the salt bridge
network is essential for the elegant reaction mechanism torealize high catalytic turnover at the sacrifice of the affinity
against NADH.
P450nor as multi-functional enzyme: P450nor exhibits
NADH-peroxidase activity, as below.
H2O2NADHH → 2H2ONAD
Here, we found a new function of P450nor, codenitrifying activity (below).
NON3 → N2ON2orNONH3 → N2OH2O

This means that P450nor forms N2O when another nitrogen donor (azide, ammonium, and so on) is supplied instead of NADH, by combining two nitrogen atoms of NO
and the other nitrogen donor. This reaction can elucidate
the phenomenon of codenitrification observed with intact
fungal cells.
Protein engineering of P450foxy: P450foxy is the fungal
counterpart of bacterial P450BM3. They resemble eachother, while a noticeable difference is observed. According
to the crystal structure of P450BM3, Phe42, Arg47, andTyr51 located at the entrance of the active site pocket, play
crucial role in binding the substrate fatty acid. Interestingly,
these amino acid residues are respectively replaced by
Leu43, Lys48, and Phe52 in P450foxy. We constructedmany mutant proteins of P450foxy in which these aminoacid residues are replaced with the corresponding aminoacids at each site. The results showed that these amino acids
are important not only in binding fatty acid substrate but
also in determining the substrate specificity.
References
1.

Su, F., Takaya, N., and Shoun, H. Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor. Biosci. Biotechnol.
Biochem. 68 (2), 473–475 (2004)
2. Su, F., Fushinobu, S., Takaya, N., and Shoun, H. Involvement of aGlu71-Arg64 couple in the access channel for NADH in cytochrome
P450nor. Biosci. Biotechnol. Biochem. 68 (5), 1156–1159 (2004)
3. Umemura, M., Su, F., Takaya, N., Shiro, Y., and Shoun, H. D88A
mutant of cytochrome P450nor provides kinetic evidence for directcomplex formation with electron donor NADH. Eur. J. Biochem. 271(14), 2887–2894 (2004)
4. Oshima, R., Fushinobu, S., Su, F., Zhang, L., Takaya, N., and Shoun,
H. Structural evidence for direct hydride transfer from NADH to cytochrome P450nor. J. Mol. Biol., in press (2004)