Communication Cite This: Biochemistry XXXX, XXX, XXX−XXX
pubs.acs.org/biochemistry
Stereochemical Course of Methyl Transfer by Cobalamin-Dependent Radical SAM Methyltransferase in Fosfomycin Biosynthesis Anna Schweifer† and Friedrich Hammerschmidt* Institute of Organic Chemistry, University of Vienna, Währingerstraße 38, A-1090 Vienna, Austria S Supporting Information *
the methyltransferase. However, the groups of Eguchi, Kuzuyama, and Nishiyama found that HEP has to be cytidylylated to give HEP-CMP (6) prior to methylation.7,8 Hydrolytic removal of CMP and epoxide ring formation by the non-heme iron HPP epoxidase finish fosfomycin biosynthesis.9 The elucidation of the methylation step has a long history concerning intermediate and mechanism (Scheme 2). Early
ABSTRACT: The methyl groups of [methyl-(S)]- and [methyl-(R)]-[methyl-D,T]-L-methionine fed to Streptomyces f radiae were incorporated into fosfomycin, which was chemically degraded to chiral AcONa. The enzymatic test gave the (S)-configuration for the chiral AcONa derived from methionine with the (S)-[D,T]methyl group (F = 31.7) and (R) for the one derived from methionine with the (R)-[D,T]methyl group (F = 83.0). The radical SAM methyltransferase transfers the methyl group of MeCbl to HEP-CMP with inversion of configuration.
F
Scheme 2. Radical SAM-Mediated Conversion of HEP-CMP (6) to HPP-CMP in Fosfomycin Biosynthesis
osfomycin [1 (see Scheme 1)]1 is a clinically used broadspectrum antibiotic2 produced by Streptomyces and
Scheme 1. Biosynthesis of Fosfomycin from Phosphoenolpyruvate
findings are that methionine is the methyl donor10 and that B12 is required11 for methylation of an unknown intermediate. Initially, phosphonoacetaldehyde (4) was reasoned to be the putative intermediate, and it was found that the methyl group is possibly transferred intact as a “CH3−” from methylcobalamin (MeCbl), although with objections.12 Van der Donk et al. found13 that the Fom3 sequence has two conserved domains, a B12-like binding one and the other one showing homology to the radical SAM protein family.14,15 On the basis of these findings, they had initially proposed that phosphonoacetalde-
Pseudomonas. It contains a P−C bond found in a small group of natural products3 and inhibits bacterial peptidoglycan biosynthesis.2 Fosfomycin biosynthesis comprises unique enzymatic transformations (Scheme 1).3 Phosphoenolpyruvate (PEP, 2) is rearranged by PEP mutase4 to phosphonopyruvate (PnPyr, 3), which is decarboxylated by a TDP-dependent decarboxylase,35 to give phosphonoacetaldehyde (PhAA, 4). This is reduced6 to 2-hydroxyethylphosphonate (HEP, 5), which was considered until very recently to be the substrate for © XXXX American Chemical Society
Received: March 3, 2018 Revised: March 21, 2018
A
DOI: 10.1021/acs.biochem.8b00264 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry hyde is first reduced to 2-hydroxyethylphosphonate (HEP, 5) undergoing radical SAM-mediated methylation with MeCbl, which was recently revised by Eguchi et al.8 The 5′deoxyadenosyl radical generated by transfer of an electron14,15 from [4Fe-4S]+ to SAM and homolytic cleavage of the C-5′− sulfur bond removes a hydrogen atom from C-2 of HEP-CMP. The ketyl radical 11 accepts a methyl radical from methylcobalamin to give 2-hydroxypropylphosphonate-CMP (7). In this Communication, we address the cryptic stereochemical course of the methyl transfer from L-methionine to ketyl radical 11, using chirally labeled methyl groups (called chiral methyl groups for the sake of simplicity). Cornforth16 and Arigoni17 and their co-workers accessed (R)- and (S)-[2D,2-T]acetic acid and determined the stereochemical course of the condensation catalyzed by malate synthase. This reaction was later used to determine the configuration and the enantiomeric excess (ee) of chiral acetic acid samples obtained by degradation of enzymatic reaction products containing a chiral methyl group. These pioneering achievements paved the way for the preparation of compounds containing chiral methyl groups and their application for the elucidation of the cryptic stereochemistry of enzymatic and chemical transformations.18−21 To elucidate the stereochemical course of the methyl transfer in fosfomycin biosynthesis, we envisaged to feed [methyl-(S)]and [methyl-(R)]-[methyl-D,T]-L-methionine to Streptomyces f radiae (Scheme 3). Degradation of the fosfomycin to chiral acetic acid and determination of its configuration and ee will allow deduction of the stereochemical course of the methyl transfer.
Scheme 4. Conversion of Fosfomycin to AcONa
temperature). Distillation furnished acetic acid isolated as sodium salt after neutralization with NaOH (63% yield). When the experiment was repeated in D2O (99.9 atom % D) under otherwise identical conditions, the sodium acetate contained 14% monodeuterated isotopomers (by 1H NMR), evidently formed via enolization during degradation. Decreasing the reaction temperature to 70 and 55 °C decreased the level of exchange of H for D to 4.5% and at best 2%, respectively, with little influence on the yield of NaOAc. Second, the conversion of nonradioactive fosfomycin in the culture broth to the diol and its isolation had to be solved. As the titer in the supernatant of the centrifuged culture broth is low (10 mg/L),9a 50 mg of fosfomycin was added before freeze-drying. The residue was dissolved in water, passed through Dowex 50WX8, H+, and eluted with water until the solution was neutral. The acidic solution was left for 48 h at 25 °C as before to allow for ring opening to the diol. The basified solution (25% NH3) was concentrated, and the diol was isolated from the residue by purification by anion exchange chromatography (Dowex 1X8, HCO 3 − , elution with Et3NH+HCO3−). The triethylammonium salt of the diol was finally converted to the sodium salt. The chirally methyl-labeled methionines had been obtained in the course of a previous project by alkylation of Lhomocysteine with (S)- and (R)-[D1,T]methyl tosylate. The former tosylate had an ee of 98% [as determined by 3H NMR, after methylation of (S)-2-methylpiperidine at N] and the latter the supposedly same ee.23 Two feeding experiments with [methyl-(S)]- and [methyl(R)]-[methyl-D,T]-L-methionine were set up with S. fradiae.9a Two flasks each contained 40 mL of medium containing 5 mg (5 μCi, 1.5 mCi/mmol) of the former and 2 mg of the latter radioactive methionine. The culture broths were combined pairwise after seeding and incubation for 3 days and then centrifuged. Nonradioactive fosfomycin (50 mg) was added to each supernatant. Workup comprised three steps: (1) conversion of [3-D,3-T]fosfomycin to 1,2-dihydroxy[3-D,3T]propylphosphonic acid and isolation, (2) Kuhn−Roth oxidation to [2-D,2-T]acetic acid, and (3) conversion of [2D,2-T]AcOH to labeled NaOAc. Malate synthase needed for the enzymatic test of labeled acetate was consistently isolated from baker’s yeast in the past. We isolated the enzyme in higher yield from yeast cells transformed with a multicopy plasmid containing the complete MLS1 gene of Saccharomyces cerevisiae.24 Transformed yeast cells express many times more malate synthase than the nontransformed cells do, and the enzyme remains cytosolic facilitating the isolation (for details, see the Supporting Information). [2-14C]Acetate was added to the two samples of isolated labeled sodium salts (18 mg, 4.87 μCi/mmol, and 20
Scheme 3. Concept for the Determination of Stereochemical Course of Methyl Transfer in Fosfomycin Biosynthesis
First, the conversion of commercial fosfomycin to acetic acid was optimized (Scheme 4). Additionally, exchange of hydrogen atoms of the methyl group was addressed, which would lead in the labeled series to partial racemization. When an aqueous solution of the disodium salt of fosfomycin was passed through Dowex 50WX8, H+, the free acid was obtained. After the solution had been left for 48 h at room temperature, the acidlabile epoxide was regioselectively ring opened. Inversion of configuration at C-2 gave (R,R)-1,2-dihydroxypropylphosphonic acid22 [(R,R)-12], which was neutralized with NaOH and subjected to Kuhn−Roth oxidation20 at 120 °C (bath B
DOI: 10.1021/acs.biochem.8b00264 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry mg, 7.59 μCi) to obtain 3H/14C ratios of 3.508 and 3.684, respectively). These samples were evalutated using the enzymatic test18−20 elaborated by the groups of Arigoni and Cornforth. The F value for the chiral acetic acid derived from [methyl-(S)]-[methyl-D,T]-L-methionine was 31.7, and that derived from the methionine with the (R)-configured chiral methyl group was 83.0. Consequently, the two samples have (S)- and (R)-configurations, respectively.18−20 The sample of acetic acid with F = 31.7 has an ee of ∼65%, and that with F = 83.0 is virtually enantiomerically pure (for calculations, see the Supporting Information).20 The lower ee of the first sample is attributed to partial racemization of the chiral methyl group during the sequence starting with the synthesis of chiral methanol and ending with the Kuhn−Roth oxidation. Our findings demonstrate that the methyl group of Lmethionine is transferred with overall net retention of configuration during fosfomycin biosynthesis, supporting the proposed reaction mechanism with two inversions. Net retention of configuraton is in line with first S-adenosylation of methionine to give SAM with retention of configuration. The direct transfer, not involving an intermediate methyl acceptor in the active site of the enzyme, of the methyl group from there to Cbl(I) (SN2-like process) should result in inversion of configuration. The attack of the ketyl radical at the MeCbl(III) represents the transfer of a methyl radical with the second inversion of configuration and the concomitant formation of Cbl(II). There are two classes of methyltransferases depending on whether they use SAM directly or indirectly via MeCbl. Enzymes of the first class transfer the methyl group from SAM to a nucleophile with inversion of configuration by an SN2-like mechanism.18 The methyltransferase of fosfomycin biosynthesis belongs to the second class of methyltransferases, specifically to the small group of class B radical SAM methyltransferases containing both a radical SAM binding domain and a cobalamin binding one.15a Other members of this growing group are involved in the biosyntheses of, for example, tert-butylglycin,21 Clorobiocin,25 phosphinothricin,26 thiostrepton,27 gentamicin G418,28,29 and thienamycin.30,31 The latter natural product biosynthesis involves two methyl transfers from methionine to introduce C-8 and C-9 of the ethyl side chain. The second one (C-9) is incorporated with net retention of configuration as proven by using methionines with chiral methyl groups as shown by Floss et al.30 They tentatively speculated that a corrin, e.g., B12, could serve as an intermediate carrier for this methyl group. It was found recently that the biosynthesis of heavily posttranslationally modified polytheonamides, potent peptide cytotoxins, comprises beside epimerases and hydroxylases two cobalamin-dependent radical SAM methyltransferases performing 17 methylations at various positions.32 Hammerschmidt reported some years ago that the deuterium atom of (R)-2-hydroxy[2-D1]ethylphosphonate is lost upon methylation and that of the (S)-enantiomer is retained as shown by feeding experiments with S. fadiae.33 This means that the pro-R hydrogen atom of HEP is removed after conversion to HEP-CMP from one side of the C-2 atom. The methyl group is then transferred to the opposite side to generate (2S)HPP-CMP, which corresponds to a formal inversion of configuration at C-2 of HEP. However, Eguchi et al. showed that by reacting HEP-CMP with reconstituted Fom3 derived from Streptomyces wedmorensis a nearly 1/1 mixture of (S)- and (R)-HPP-CMP was surprisingly generated.8 These seemingly
conflicting results may be explained as a result of (1) the utilization of the reconstituted enzyme from S. wedmorensis versus feeding experiments with S. f radiae or (2) the fact that the (R)-2-hydroxypropylphosphonate possibly also formed in S. f radiae is converted by the HPP epoxidase9d only present in the cell system to 2-oxopropylphosphonate. Liu et al. proved very recently that the reaction catalyzed by GenK, a cobalamindependent radical SAM methyltransferase in the biosynthetic pathway of gentamicin, proceeds with retention of configuration at the C(6′)H2OH group.29 In summary, we have unraveled the cryptic stereochemical course for the methyl transfer from L-methionine to the intermediate 2-hydroxyethylphosphonate-CMP of fosfomycin biosynthesis. The data are consistent with transfer in two steps, from SAM to Cbl(I) and then from the MeCbl(III) to a carbon atom with net retention of configuration. Consequently, the involved radical SAM methyltransferase effects the methyl transfer from MeCbl(III) with inversion of configuration. No study has really shown if it is SAM or MeCbl that donates the methyl group. This study (as well as the work by Floss on thienamycin) provides evidence that is consistent with the methyl group indeed being transferred from SAM to cobalamin.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00264. Experimental procedures and characterization data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Friedrich Hammerschmidt: 0000-0003-2193-1405 Funding
The authors thank the Austrian Science Fund (Grant P14985− N03) for financial support. Notes
The authors declare no competing financial interest. † Deceased.
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ACKNOWLEDGMENTS The authors thank M. Cichna-Markl for using the radiochemistry laboratory at the Department of Analytical Chemistry and A. Hartig and F. Koller (Max F. Perutz Laboratories, Department of Biochemistry and Cell Biology, University of Vienna) for transformed yeast cells and help with isolation of malate synthase.
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REFERENCES
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