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C-Methylation Catalyzed by Fom3, a Cobalamin-dependent Radical SAM Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration Shusuke Sato, Fumitaka Kudo, Tomohisa Kuzuyama, Friedrich Hammerschmidt, and Tadashi Eguchi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00614 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018
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Biochemistry
C-Methylation Catalyzed by Fom3, a Cobalamin-dependent Radical SAM Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration Shusuke Sato,† Fumitaka Kudo,†,* Tomohisa Kuzuyama,‡,§ Friedrich Hammerschmidt,§ and Tadashi Eguchi†,* †Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan ‡Biotechnology Research Center, §Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan §Institute of Organic Chemistry, University of Vienna, Währingerstraße 38, A-1090 Vienna, Austria Supporting Information
adenosyl-L-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In the present study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be (S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is (S)selective. Further, Fom3 reaction was carried out with (S)-[2-2H1]HEP-CMP and (R)-[2-2H1]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. LC-ESI-MS analysis of the 5'-deoxyadenosine produced showed that the 2H atom of (R)-[2-2H1]HEP-CMP was incorporated into 5'-deoxyadenosine, but that from (S)-[2-2H1]HEPCMP was not. Retention of the 2H atom of (S)-[22 H1]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located at the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.
thyl oxirane. The methyl group of fosfomycin is introduced by a radical S-adenosyl-L-methionine (SAM) enzyme, Fom3.2-4 Radical SAM enzymes have an ironsulfur cluster at the active site and the reduced form cleaves the carbon–sulfur bond of SAM to reductively generate the 5'-deoxyadenosyl radical (5'-dA•) as a radical initiator.5 Fom3 is annotated as a cobalamindependent radical SAM methyltransferase (RSMT) which catalyzes C-methylation by transfer of the methyl group on methylcobalamin (MeCbl) to the radical intermediate.6 Although the substrate of Fom3 had been proposed for a long time to be 2-hydroxyethylphosphonate (HEP),7,8 cytidylylated 2-hydroxyethyphosphonate (HEP-CMP) was found to be an true substrate because the N-terminal domain of Fom1, which is responsible for the C–P bond formation, catalyzes the cytidylylation of HEP with cytidine triphosphate (CTP) to give HEPCMP.9 In fact, Fom3 converts HEP-CMP to cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in the presence of SAM, MeCbl, methyl viologen, NADH and dithiothreitol (Scheme 1).10 The production of 5'deoxyadenosine (5'-dA) was also observed, indicating that 5'-dA• abstracted the hydrogen atom of HEP-CMP. In the presence of HEP instead of HEP-CMP, the production of 5'-dA was not detected, confirming that HEP is not a substrate of Fom3. Thus, the CMP moiety of HEP-CMP appears to be critical for substrate recognition by Fom3.
Fosfomycin (Scheme 1) is a clinically used antibiotic produced by Streptomyces and Pseudomonas.1 A structural feature of fosfomycin is the characteristic phosphonate group that is attached to the C1 position of me-
Previous studies showed that the chirally isotope-labeled methyl group of L-methionine was incorporated into fosfomycin with overall net retention of configuration and indicated that two inversions occur during the methyl transfer reaction.11 Thus, the methyl group on MeCbl that is generated from SAM by
ABSTRACT: Fom3, a cobalamin-dependent radical (S)-
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Scheme 1. Fosfomycin Biosynthetic Pathway O
O
Fom1 Fom2 FomC
HO P HO O OH
O Phosphoenolepyruvate
CH3
O
HO P OH NH2 O Fom1 Fom3 O N HO P OH HO P O N O HO O O 2-HydroxyethylOH OH phosphonate (HEP) HEP-CMP
HO
P
O P O HO O
OH NH2 N O
N
CH3
O
FomD
HO P HO
O
OH
Fom4
O HO P HO H
(S)-2-Hydroxypropylphosphonate ((S)-HPP)
OH OH HPP-CMP
CH3 O
H
Fosfomycin
a SN2 like reaction is transferred to the C2 position of HEP-CMP with inversion of configuration. Previous studies also showed that feeding of [2,22 H2]HEP and (S)-[2-2H1]HEP to Streptomyces fradiae resulted in uptake of deuterium into fosfomycin, whereas no deuterium was retained in fosfomycin when (R)[2-2H1]HEP was fed.12,13 Therefore, Fom3 has been supposed to abstract the pro-R hydrogen atom at C2 of HEP-CMP to afford (S)-HPP-CMP, because only (S)HPP is converted to fosfomycin by the non-heme irondependent peroxidase Fom4.14,15 Thus, the methylation of HEP-CMP by Fom3 seems to occur through inversion of configuration at the C2 position.8 However, our previous experiments showed that the Fom3 reaction product from HEP-CMP was a diastereomeric mixture of HPP-CMP.10 In this study, we reinvestigated the stereochemistry of the C-methylation with deuterium labeled HEP-CMP.
of (S)- and (R)-HPP-CMP (Figure 2). The chelation of Cu2+ by the 2-OH group and phosphonic acid of HPPCMP would change the conformation of the two diastereomers leading to the good separation. HPLC analysis of the Fom3 reaction product HPP-CMP using the same chromatographic conditions revealed that only (S)HPP-CMP was produced (Figures 2 and S4). Therefore, it was found that the C-methylation catalyzed by Fom3 is (S)-selective.
At first, we carried out the Fom3 reaction with [2,22 H2]HEP-CMP, which was prepared from [2,2-2H2]HEP and CTP using Fom1,9 to confirm that the H atom at C2 of HEP-CMP is abstracted and incorporated into 5'-dA. Liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS) of the 5'-dA produced from SAM and [2,2-2H2]HEP-CMP showed a peak at m/z = 253, indicating that one deuterium atom of [2,22 H2]HEP-CMP was incorporated into the 5'-dA (Figure 1A). 1H and 2H NMR of the isolated 5'-dA showed that the deuterium atom was incorporated into the C5' position (Figure S1). 1H NMR of the isolated HPP-CMP showed that one deuterium atom at the C2 position of [2,2-2H2]HEP-CMP was retained in HPP-CMP, because the signal of the C2-methyl group at 1.09 ppm was a singlet (Figure S2 and S3). However, a doublet at 1.14 ppm in this 1H-NMR spectrum seems to be derived from a methyl group of a contamination in the enzyme solution. A careful inspection of our previous NMR spectra of the Fom3 reaction products and authentic HPP-CMP revealed that the same impurity was present in the solutions. Therefore, it was found that we could not distinguish the methyl signals of (S)- and (R)-HPP-CMP by NMR.
c
Then, we examined the HPLC separation of a mixture of (S)- and (R)-HPP-CMP. Chiral ligand exchange chromatography with a N,S-dioctyl-D-penicillamine octadecyl coated silica gel column using a copper(II)-containing solution as the mobile phase resulted in clear separation
A
a
b
B 5'-dA [M+H]+ m/z 252
f
5'-dA [M+H]+ m/z 252
g [5'-2H]5'-dA [M+H]+ m/z 253
e
i
5'-dA [M+H]+ m/z 252 251
252
HPP-CMP [M-H]m/z 444 [2-2H]HPP-CMP [M-H]m/z 445
h
[5'-2H]5'-dA [M+H]+ m/z 253
d
HPP-CMP [M-H]m/z 444
HPP-CMP [M-H]m/z 444 [2-2H]HPP-CMP [M-H]m/z 445
j 253 m/z
254
255
442
443
444 445 m/z
446
447
Figure 1. LC-ESI-MS analysis of Fom3 reaction products from deuterated substrates. (A) MS spectra of the generated 5'-dA in the positive scanning mode at retention time of 12 min. (a) Authentic sample of 5'-dA, (b) 5'-dA from HEPCMP, (c) 5'-dA from [2,2-2H2]HEP-CMP, (d) 5'-dA from (R)-[2-2H1]HEP-CMP, (e) 5'-dA from (S)-[2-2H1]HEP-CMP. (B) MS spectra of the methylation product in the negative scanning mode at retention time of 13 min. (f) Authentic sample of HPP-CMP, (g) HPP-CMP from HEP-CMP, (h) HPP-CMP from [2,2-2H2]HEP-CMP, (i) HPP-CMP from (R)-[2-2H1]HEP-CMP, (j) HPP-CMP from (S)-[2-2H1]HEPCMP.
Next, to verify the stereochemistry during the abstraction of a hydrogen atom from C2 of HEP-CMP, the Fom3 reaction was carried out with (S)- and (R)-[22 H1]HEP-CMP, which were enzymatically synthesized from (S)-[2-2H1]HEP and (R)-[2-2H1]HEP, respectively. LC-ESI-MS analysis of the 5'-dA produced from (S)-[22 H1]HEP-CMP showed a peak at m/z = 252, corresponding to [M+H]+ of 5'-dA, whereas m/z = 253 was observed from (R)-[2-2H1]HEP-CMP, indicating the 2H atom was incorporated into 5'-dA (Figure 1A). MS spec-
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tra of the methylation product of the substrate from (R)[2-2H1]HEP-CMP showed at peak at m/z = 444, corresponding to [M-H]− of HPP-CMP, whereas m/z = 445 was observed from (S)-[2-2H1]HEP-CMP, indicating the 2 H atom was retained in HPP-CMP (Figure 1B). These results suggest that 5'-dA• abstracts the 2H atom of (R)[2-2H1]HEP-CMP to afford deuterium-labeled 5'-dA, whereas 5'-dA• abstracts the 1H atom of (S)-[2-2H1]HEPCMP and the 2H atom of (S)-[2-2H1]HEP-CMP remained in the methylation product. Hence, the pro-R hydrogen atom at C2 of HEP-CMP is selectively abstracted by 5'dA• to afford the substrate radical intermediate. Moreover, we carried out kinetic experiments using deuterium labeled substrates (Figure S5). The reaction rate with [2,2-2H2]- or (R)-[2-2H1]HEP-CMP was lower than that with unlabeled or (S)-[2-2H1]HEP-CMP, supporting the stereoselective abstraction of the pro-R hydrogen atom at the C2 position.
achieve retention of configuration. However, at the active site of Fom3, the HEP-CMP substrate is probably sandwiched between two cofactors, SAM and MeCbl. GenD1 catalyzes C-methylation at C4'' of gentamicin A in gentamicin biosynthesis, also with inversion of configuration.17 Therefore, Fom3 and GenD1 can be classified as the same enzyme subtype. e-
NH2 N
H
[4Fe-4S]+
H2C
[4Fe-4S]2+
N
N
O
N
HO OH
5'-dA
SAM
5'-dA
HO
P
O H3C H
O
O HS HR OH
HO
O-CMP
H
P
HO
OH CH3
HEP-CMP NH2 N
O S
HO
O
e-
N
CoI
NH2
NH2 HO OH
CH3 CoIIl
300
a
250 200
b
150
c
100 50
d 0 4
5
6 7 8 Retention time [min]
9
10
Figure 2. Chiral ligand exchange chromatography of (S)- and (R)-HPP-CMP. (a) Authentic sample of a diastereomeric mixture of HPP-CMP, (b) authentic sample of (S)-HPP-CMP (≥90% ee), (c) authentic sample of (R)-HPP-CMP (≥99% ee), (d) isolated Fom3 methylation product. The proposed reaction mechanism of the C-methylation catalyzed by Fom3 is shown in Figure 3. The 5'-dA• generated from SAM stereoselectively abstracts the proR hydrogen atom at the C2 position of HEP-CMP to form 5'-dA and the substrate radical. The methyl group on MeCbl is transferred to the substrate radical from the opposite side of the abstracted hydrogen atom to afford (S)-HPP-CMP. So, the C-methylation catalyzed by Fom3 proceeds with inversion of configuration. Recently, it has been reported that the C-methylation catalyzed by GenK, which is a cobalamin-dependent RSMT involved in gentamicin biosynthesis, proceeds with retention of configuration.16 At the active site of GenK, the iron-sulfur cluster and MeCbl are proposed to be arranged in parallel to the substrate in order to
OH
CoIl
CoIIl
CH3
O
= Methylcobalamin (Vitamin B12)
N
S
HO
350
(S)
(S)-HPP-CMP
N
N
P
O-CMP
O-CMP
SAH
AU (280 nm) [mV]
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Biochemistry
O
N
N N
NH2 HO OH
SAM
Figure 3. Inversion of configuration in C-methylation catalyzed by Fom3.
In summary, we have demonstrated that the Cmethylation catalyzed by Fom3 proceeds with inversion of configuration at the C2 position of HEP-CMP. Although both Fom3 and GenK methylate the sp3-carbon of a primary alcohol, the stereochemical course of the methyl transfer is different. The reasons for these differences probably lie in the structures of the active sites of cobalamin-dependent RSMTs. The only known crystal structure of a cobalamin-dependent radical SAM enzyme, OxsB, which catalyzes the formation of the fourmembered ring of oxetanocin A, revealed that SAM and cobalamin interplay during the radical reaction.18 Since the sequence of OxsB is only 11–12% identical to that of Fom3 or GenK, it is hard to discuss the differences between Fom3 and GenK based on the structure of OxsB. Further analysis, including structural analysis, is required to understand the reaction mechanism(s) of the cobalamin-dependent radical SAM methyltransferases. ASSOCIATED CONTENT Supporting Information is available free of charge on the ACS Publications website. Experimental details, supporting tables and figures
AUTHOR INFORMATION Corresponding Authors
*
[email protected], *
[email protected] ORCID
Fumitaka Kudo: 0000-0002-4788-0063
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Tomohisa Kuzuyama: 0000-0002-7221-5858 Friedrich Hammerschmidt: 0000-0003-2193-1405 Tadashi Eguchi: 0000-0002-7830-7104 Author Contributions
SS, FK, TK, FH and TE designed the research; SS performed the experiments; SS, FK and TE analyzed data and wrote the manuscript; all authors approved the final version of the manuscript. Notes The authors declare no competing financial interest. Acknowledgment This work was supported in part by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) 18H02095, Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research in Innovative Areas (16H06451 to T.E. and 16H06453 to T.K.), and the Japan Society for the Promotion of Science A3 Foresight Program.
REFERENCES (1) Horsman, G. P., Zechel, D. L. (2017) Phosphonate Biochemistry. Chem. Rev. 117, 5704-5783. (2) Hidaka, T., Goda, M., Kuzuyama, T., Takei, N., Hidaka, M., Seto, H. (1995) Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis. Mol. Gen. Genet. 249, 274280. (3) Woodyer, R. D., Shao, Z., Thomas, P. M., Kelleher, N. L., Blodgett, J. A., Metcalf, W. W., van der Donk, W. A., Zhao, H. (2006) Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster. Chem. Biol. 13, 1171-1182. (4) Allen, K. D., Wang, S. C. (2014) Initial characterization of Fom3 from Streptomyces wedmorensis: the methyltransferase in fosfomycin biosynthesis. Arch. Biochem. Biophys. 543, 67-73. (5) Broderick, J. B., Duffus, B. R., Duschene, K. S., Shepard, E. M. (2014) Radical S-adenosylmethionine enzymes. Chem. Rev. 114, 4229-4317. (6) Bauerle, M. R., Schwalm, E. L., Booker, S. J. (2015) Mechanistic diversity of radical S-adenosylmethionine (SAM)-dependent methylation. J. Biol. Chem. 290, 3995-4002. (7) Hammerschmidt, F. (1994) Incorporation of L-[methyl-2H3]methionine and 2-[hydroxyl-18O]hydroxyethylphosphonic acid into fosfomycin in Streptomyces fradiae - an unusual methyl transfer. Angew. Chem. Int. Ed. Engl. 33, 341-342.
(8) Woodyer, R. D., Li, G., Zhao, H., van der Donk, W. A. (2007) New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin. Chem. Commun. 359-361. (9) Cho, S.-H., Kim, S.-Y., Tomita, T., Shiraishi, T., Park, J. S., Sato, S., Kudo, F., Eguchi, T., Funa, N., Nishiyama, M., Kuzuyama, T. (2017) Fosfomycin biosynthesis via transient cytidylylation of 2hydroxyethylphosphonate by the bifunctional Fom1 enzyme. ACS Chem. Biol. 121, 2209-2215. (10) Sato, S., Kudo, F., Kim, S.-Y., Kuzuyama, T., and Eguchi, T. (2017) Methylcobalamin-dependent radical SAM C-methyltransferase Fom3 recognizes cytidylyl-2-hydroxyethylphosphonate and catalyzes the nonstereoselective C-methylation in fosfomycin biosynthesis. Biochemistry 56, 3519-3522. (11) Schweifer, A., Hammerschmidt, F. (2018) Stereochemical course of methyl transfer by cobalamin-dependent radical SAM methyltransferase in fosfomycin biosynthesis. Biochemistry 57, 2069-2073. (12) Hammerschmidt, F., and Kaehlig, H. (1991) Biosynthesis of natural products with a phosphorus-carbon bond. VII. Synthesis of [2,2-2H2]-, (R)and (S)-[1-2H1](2[1,1-2H2]-, hydroxyethyl)phosphonic acid and (R,S)-[1-2H1](1,2dihydroxyethyl)phosphonic acid and incorporation studies into fosfomycin in Streptomyces fradiae. J. Org. Chem. 56, 2364-2370. (13) Hammerschmidt, F. (1992) Biosynthesis of natural products with a phosphorus-carbon bond. IX. Synthesis and incorporation of (S)and (R)-2-hydroxy-[2-2H1]ethylphosphonic acid into fosfomycin by Streptomyces fradiae. Liebigs Ann. Chem. 6, 553-557. (14) Liu, P., Murakami, K., Seki, T., He, X., Yeung, S.-M., Kuzuyama, T., Seto, H., Liu, H.-w. (2001) Protein Purification and function assignment of the epxidase catalyzing the formation of fosfomycin J. Am. Chem. Soc. 123, 4619-4620. (15) Zhao, Z., Liu, P., Murakami, K., Kuzuyama, T., Seto, H., Liu, H.-w. (2002) Mechanistic studies of HPP epoxidase: configuration of the substrate governs its enzymatic fate. Angew. Chem. Int. Ed. Engl. 41, 4529-4532. (16) Kim, H. J., Liu, Y. N., McCarty, R. M., and Liu, H.-w. (2017) Reaction catalyzed by GenK, a cobalamin-dependent radical Sadenosyl-L-methionine methyltransferase in the biosynthetic pathway of gentamicin, proceeds with retention of configuration. J. Am. Chem. Soc. 139, 16084-16087. (17) Huang, C., Huang, F., Moison, E., Guo, J., Jian, X., Duan, X., Deng, Z., Leadlay, P. L., and Sun, Y. (2015) Delineating the biosynthesis of gentamicin X2, the common precursor of the gentamicin C antibiotic complex. Chem. Biol. 22, 251-261. (18) Bridwell-Rabb, J., Zhong, A., Sun, H. G., Drennan, C. L., Liu, H.-w. (2017) A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis. Nature 544, 322-326.
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Biochemistry
Insert Table of Contents artwork here cobalamin CoIIl
H2N
HO
Fom3
O
N O
HO
[4Fe-4S]2+
N OH
N N
5'-dA•
H2N
OH
P
HO
O-
O
P O-
NH2 H H2C HO
N N
O
OH
[4Fe-4S]2+
H2N
O
N
N O
O
Fom3
CoII
N
CH3 H
OH
HEP-CMP NH2
H2C
O
P P O O OO-
HR
O N
O
HS
CoIIl
N
CH3
O O
O N
CH3
OH OH
Fom3
H HO
(S)
O
O
P P O O OO-
O
OH OH
(S)-HPP-CMP
NH2
N
H H2C
N
5'-dA
HO
N N
O
OH
N N
5'-dA
[4Fe-4S]2+
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