Methylcobalamin-Dependent Radical SAM C

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Methylcobalamin-dependent Radical SAM C-Methyltransferase Fom3 Recognizes Cytidylyl-2-hydroxyethylphosphonate and Catalyzes the Non-stereoselective C-Methylation in Fosfomycin Biosynthesis Shusuke Sato, Fumitaka Kudo, Seung-Young Kim, Tomohisa Kuzuyama, and Tadashi Eguchi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00472 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017

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Biochemistry

Methylcobalamin-dependent Radical SAM CMethyltransferase Fom3 Recognizes Cytidylyl-2hydroxyethylphosphonate and Catalyzes the Nonstereoselective C-Methylation in Fosfomycin Biosynthesis Shusuke Sato,† Fumitaka Kudo,*† Seung-Young Kim,‡ Tomohisa Kuzuyama,‡ and Tadashi Eguchi*† † ‡

Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Supporting Information Placeholder ABSTRACT: A methylcobalamin (MeCbl)-dependent radical S-adenosyl-L-methionine (SAM) methyltransferase Fom3 was found to catalyze the C-methylation of cytidylyl-2hydroxyethylphosphonate (HEP-CMP) to give cytidylyl-2hydroxypropylphosphonate (HPP-CMP), although it was originally proposed to catalyze the C-methylation of 2hydroxyethylphosphonate (HEP) to give 2hydroxypropylphosphonate (HPP) in the biosynthesis of a unique C–P bond containing antibiotic fosfomycin in Streptomyces. Unexpectedly, the Fom3 reaction product from HEP-CMP was almost a 1:1 diastereomeric mixture of HPPCMP, indicating that the C-methylation is not stereoselective. Presumably, only the CMP moiety of HEP-CMP is critical for substrate recognition, whereas the enzyme does not fix the 2hydroxy group of the substrate and either of the prochiral hydrogen atoms at the C2 position can be abstracted by the 5'deoxyadenosyl radical generated from SAM to form the substrate radical intermediates, which react with MeCbl to afford the corresponding products. This strict substrate recognition mechanism with no stereoselectivity of a MeCbldependent radical SAM methyltransferase is remarkable in natural product biosynthetic chemistry, because such a hidden clue for selective substrate recognition is likely to be found in the other biosynthetic pathways.

Fosfomycin (Scheme 1) is a clinically approved broadspectrum antibiotic with a characteristic C–P bond.1-3 Intriguingly, the producer microorganisms Streptomyces and Pseudomonas use different biosynthetic pathways even though the first C–P bond forming enzyme and the last epoxide-forming enzyme are shared in both genera of bacteria.4-5 In Streptomyces, five genes were identified to be involved in the biosynthesis of fosfomycin by genetic analysis and in vitro enzymatic analysis with recombinant enzymes (Scheme 1, path A).6-8 Initially, phosphoenolpyruvate (PEP) is converted to phosphonopyruvate (PnPy) by PEP mutase Fom1, which is conserved in both Streptomyces and Pseudomonas.5, 9 PnPy is then decarboxylated by a

thiamin-dependent decarboxylase Fom2 to produce 10-11 phosphonoacetaldehyde (PnAA).6, The decarboxylation reaction presumably drives the unfavorable PEP mutase reaction forward. PnAA is then converted to 2-hydroxyethylphosphonate (HEP) by an alcohol dehydrogenase FomC.12-13 HEP is proposed to be methylated to generate 2-hydroxypropylphosphonate (HPP) by a methylcobalamin (MeCbl)-dependent radical S-adenosyl-L-methionine (SAM) methyltransferase Fom3.13-16 It was revealed that methylcobalamin is the direct methyl donor by previous feeding experiment.15 In the final step, (S)-HPP is oxidized to form an epoxide, completing the biosynthesis of fosfomycin. This epoxidation step is catalyzed by a non-heme-iron peroxidase Fom4 that is also conserved in all fosfomycin producer strains that have been characterized.4, 17-18

Scheme 1. Two plausible biosynthetic pathways for fosfomycin in Streptomyces are the (A) previously proposed pathway and (B) the newly proposed pathway presented in this study.

Recently, it was found that the N-terminal domain of Fom1 derived from Streptomyces wedmorensis is a cytidylyltransferase (Fom1 CyTase domain), which catalyzes the cytidylylation of HEP with CTP to give cytidylyl-

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HEP (HEP-CMP).19 Further, the feeding of HPP and not HEP to a deletion mutant of the fom1 gene recovered the production of fosfomycin. Therefore, the cytidylylation reaction of HEP appears to be involved in fosfomycin biosynthesis in Streptomyces. Thus, the generated HEPCMP is hypothesized to be methylated by Fom3 to afford cytidylyl-HPP (HPP-CMP) (Scheme 1, path B). In the present study, we carried out enzymatic analysis of Fom3 with HEP-CMP as the potential substrate. Several MeCbl-dependent radical SAM methyltransferases including GenK20, GenD121, ThnK22, PoyC23, and CysS24 have been characterized to use 5'-deoxyadenosyl radical from SAM during the methylation. Whilst, TsrM is an unusual cobalamin dependent methyltransferase with radical SAM sequence motif and catalyzes the Cmethylation without the formation of 5'deoxyadenosine.25-26 Thus, precise functional analysis of Fom3 should provide additional valuable knowledge about this enzyme family.27-28 The fom3 gene derived from S. wedmorensis was cloned into a pET28 vector and expressed in Escherichia coli (E. coli). The suf gene cluster derived from E. coli was co-expressed to obtain the active iron sulfur cluster containing Fom3 (See experimental detail in Supporting Information).29-30 The E. coli cells harboring the expressed protein were disrupted in a glove box and the soluble protein fraction was purified by affinity chromatography with TALON® resin (Figure S1). The obtained protein solution was incubated in the presence of FeSO4(NH4)2SO4, FeCl3 and Na2S to reconstitute the iron sulfur cluster. The presence of the oxidized [4Fe4S]2+ cluster was confirmed by UV-visible spectroscopic analysis by observing the characteristic absorption at 420 nm (Figure S2). After treatment with sodium dithionite, the bleached absorption at 420 nm confirmed that the iron sulfur cluster could be reduced to its active form [4Fe-4S]+1 (Figure S2). It was confirmed that Fom3 has a single [4Fe-4S] cluster.14 The reconstituted Fom3 was reacted with HEP-CMP, which was prepared from HEP and CTP with Fom1 CyTase domain (See experimental detail in Supporting Information). The reaction product was analyzed by HPLC and was compared with authentic (S)-HPP-CMP, which was also prepared from (S)-HPP and CTP with Fom1 CyTase domain. When 1 mM of HEP-CMP was incubated in the presence of 4 mM SAM, 0.1 mM MeCbl, 1 mM methylviologen (MV), 4 mM NADH and 10 mM dithiothreitol (DTT) with 2 µM Fom3 in a shaded tube to prevent non-enzymatic decomposition of MeCbl at 28 °C, the production of HPP-CMP was clearly observed (Figure 1A). When we used dithionite or flavodoxin/flavodoxinreductase/NADH as reducing reagents, no detectable amount of product was observed (data not shown). The production of 5'-deoxyadenosine (5'-dA) was also observed to indicate that the 5'deoxyadenosyl radical was generated to abstract the hydrogen atom of the substrate HEP-CMP (Figure 1B).

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The production of S-adenosyl-L-homocysteine (SAH) was also observed, indicating that MeCbl is regenerated from SAM for the catalytic cycle (Figure S3). The requirement of DTT supports the regeneration of MeCbl because the generated cob(II)alamin is reduced by DTT and the resulting cob(I)alamin reacts with SAM to regenerate MeCbl.31-32 The production ratio of HPPCMP, 5'-dA, and SAH was almost equal, suggesting that two equivalents of SAM are used for the C-methylation of HEP-CMP to afford HPP-CMP (Figure S3). In the presence of “HEP” instead of HEP-CMP, the production of 5'-dA was not detected, confirming that HEP is not an appropriate substrate of Fom3 (Figure 1B). Allen and Wang reported a controversial result that a small amount of HPP was generated from HEP with the reconstituted Fom3 by showing the 31P-NMR of the reaction product.14 Their preliminary observation seems thus a minor activity with unfavorable substrate HEP, because the enzymatic activity was very low. Thus, the CMP moiety of HEP-CMP was found to be critical for substrate recognition by Fom3. HEP-CMP is the likely biosynthetic intermediate in the fosfomycin biosynthetic pathway. A B HEP-CMP

HPP-CMP

a

5'-dA

a b

b

c

c d e

d 3

5 4 retention time [min]

6

30

31 32 retention time [min]

Figure 1. Fom3 reaction with HEP-CMP or HEP. (A) HPLC traces monitored at 280 nm showed the methylation product: (a) authentic (S)-HPP-CMP, (b) Fom3 reaction, (c) without Fom3 and (d) without DTT. (B) HPLC traces monitored at 254 nm showed the production of 5'-dA: (a) authentic 5'-dA, (b) Fom3 reaction, (c) without Fom3, (d) without DTT and (e) “HEP” instead of HEP-CMP.

The Fom3 reaction product was isolated by anion exchange chromatography followed by preparative HPLC to confirm the chemical structure with LC-ESIMS and NMR. The LC-ESI-MS analysis (negative mode) of the isolated product gave an m/z = 444, which corresponds to [M-H]– of HPP-CMP (Figure S4). In the 1 H-NMR spectrum of the product, two sets of doublet methyl signals were observed around 1.15 ppm to indicate that Fom3 produced a mixture of two diastereomers (Figure 2, Figure S5). 1H-NMR spectra of

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Biochemistry

authentic (S)- and (R)-HPP-CMP clearly confirmed that Fom3 produced both diastereomers (Figure S6-S14). The epimerization of (S)-HPP-CMP was not observed in the same enzymatic reaction conditions with active Fom3 (Figure S15). The ratio of the (S)- and (R)-HPPCMP in the Fom3 reaction product was almost equal, thereby revealing that there is no stereoselectivity during the C-methylation. This enzymatic property is quite characteristic against the stereoselective C-methylation catalyzed by other MeCbl-dependent radical SAM enzymes, such as GenK in the C-methylation of gentamicin X2.20 Detailed biochemical analysis of Fom3 including protein structure determination is required to understand this unusual stereoselectivity. Steady state kinetic analysis using the Michaelis-Menten equation was performed by detecting the production of 5'-dA (Figure S16). The KM value for HEP-CMP was estimated to be 113 ± 15 µM. The kcat value was estimated to be 0.89 ± 0.03 hour–1, which is similar to that of GenK (1.2 hour-1).20 It is noted that excess amount of MeCbl against Fom3 was required for the steady state kinetic analysis (Figure S17). This suggests that the regeneration of MeCbl occurs outside of Fom3 and the formation rate is fast enough against Cmethylation. These data support that Fom3 is a typical MeCbl-dependent radical SAM C-methyltransferase, which strongly associates with cobalamin. The release of the generated cob(II)alamin may be the rate-limiting step, although additional experiments with this technically challenging enzyme are necessary to draw this conclusion. The reaction mechanism of Fom3 seems to be similar to those of GenK20 and CysS24 (Figure 3). The 5'deoxyadenosyl radical (5'-dA•) generated from the

reductive cleavage of SAM abstracts one of the hydrogen atoms at C2 of HEP-CMP to generate the substrate radical intermediate, which reacts with the methyl group on MeCbl at the opposite side from SAM and the 4Fe-4S cluster to give (S)- and (R)-HPP-CMP. Fom3 might not be able to distinguish between the two diastereotopic hydrogen atoms at C2 of HEP-CMP to generate the radical intermediate. Only the CMP moiety of HEP-CMP seems critical for recognition, whilst Fom3 presumably does not fix the 2-OH group of the HEP moiety. The oxidized [4Fe-4S]2+ cluster is reduced by the MV/NADH reducing system under the examined enzymatic conditions. The generated cob(II)alamin is reduced by DTT to cob(I)alamin, which is methylated in the presence of SAM to regenerate MeCbl. The Fom3 reaction product (S)-HPP-CMP from HEPCMP appears to be the likely biosynthetic intermediate in the fosfomycin biosynthetic pathway. Since CMPHPP was not oxidized by Fom4 presumably due to the existence of the CMP moiety, the phosphoryl phosphate of (S)-HPP-CMP must be hydrolyzed to give (S)-HPP, which is then oxidized by Fom4 to produce fosfomycin. A candidate enzyme responsible for the reaction is FomD, which is encoded in the middle of the biosynthetic gene cluster and transcriptionally coupled to other genes.6 FomD showed 34% identity to a putative phosphatase SCO5041 (Sc4828) in Streptomyces coelicolor A3(2), which is a DUF402 domain-containing protein and the crystal structure with guanosine 5'-[α,β-methylene]diphosphonic acid has been solved (PDB ID: 3EXM). Furthermore, a homologous enzyme SA1684 in Staphylococcus aureus was recently characterized as a virulence factor and catalyzes the hydrolysis of nucleotide diphosphates.33

Figure 2. 1H-NMR (400 MHz, D2O) of Fom3 reaction product HPP-CMP from HEP-CMP. (a) Isolated Fom3 reaction product, (b) authentic (S)-HPP-CMP, (c) authentic (R)-HPP-CMP, and (d) a 2:1 mixture of authentic (S)- and (R)-HPP-CMP.

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FomD is thus hypothesized to work as a hydrolase of (S)-HPP-CMP to afford (S)-HPP. The functional analysis of FomD is underway in our laboratory. The formation of (R)-HPP-CMP by Fom3 was unexpected and appears superfluous for the producer strain even if a certain hydrolase can hydrolyze (R)HPP-CMP, because Fom4 converts only (S)-HPP to fosfomycin and (R)-HPP is oxidized to 2oxopropylphosphonate (OPP).34-36 The unnecessarily generated OPP might be stereoselectively reduced by a particular reductase like Psf3, which catalyzes the stereoselective reduction of OPP to give (S)-HPP in the fosfomycin biosynthesis by Pseudomonas.4 The physiological function for this unnecessary enzymatic activity of Fom3 is an additional topic to be resolved. In conclusion, we characterized the function of a unique MeCbl-dependent radical SAM C-methyltransferase Fom3 that strictly recognizes HEP-CMP as a substrate to afford (S)- and (R)-HPP-CMP. It is noteworthy that the existence of the nucleotide moiety for the selective substrate recognition is critical rather than the stereoselectivity of the C-methylation in the Fom3 reaction. Related cytidylylated biosynthetic intermediates are involved in the biosynthesis of a phosphonate antibiotic FR-900098.37 It is also known that the hydrolysis of CMP–FR-900098 is the last biosynthetic step. Thus, the naturally occurring property of substrate selectivity would allow feasible biosynthesis of target bioactive molecules by adding a clue for substrate recognition in living systems. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxx Experimental details, supporting tables and, supporting figures.

AUTHOR INFORMATION Corresponding Author *[email protected], *[email protected]

Notes The authors declare no competing financial interests.

Author Contributions SS, FK, TK and TE designed the research; SS and SYK performed the experiments; SS, FK and TE analyzed data and wrote the manuscript; all authors approved the final version of the manuscript.

Funding Sources This work was supported in part by JSPS KAKENHI (16H06451, 15H03834 to TE and 16H06453 to TK). SYK was supported by a Postdoctoral Fellowship (23 · 01399) from JSPS.

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