Evidence for a 1,3-Dipolar Cyclo-addition Mechanism in the

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Evidence for a 1,3-Dipolar Cyclo-addition Mechanism in the Decarboxylation of Phenylacrylic Acids Catalyzed by Ferulic Acid Decarboxylase Kyle L. Ferguson,† Joseph D. Eschweiler,† Brandon T. Ruotolo,† and E. Neil G. Marsh*,†,‡ †

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States

‡

S Supporting Information *

ABSTRACT: Ferulic acid decarboxylase catalyzes the decarboxylation of phenylacrylic acid using a newly identified cofactor, prenylated flavin mononucleotide (prFMN). The proposed mechanism involves the formation of a putative pentacyclic intermediate formed by a 1,3 dipolar cyclo-addition of prFMN with the α−β double bond of the substrate, which serves to activate the substrate toward decarboxylation. However, enzymecatalyzed 1,3 dipolar cyclo-additions are unprecedented and other mechanisms are plausible. Here we describe the use of a mechanism-based inhibitor, 2-fluoro-2-nitrovinylbenzene, to trap the putative cyclo-addition intermediate, thereby demonstrating that prFMN can function as a dipole in a 1,3 dipolar cyclo-addition reaction as the initial step in a novel type of enzymatic reaction.

Figure 1. prFMN is biosynthesized from reduced FMN and dimethylallyl phosphate (DMAP) catalyzed by PAD1 (yeast) or UbiX (E. coli) followed by oxidation.

ichia coli, this reaction is catalyzed by the prFMN-dependent enzyme UbiD and the cofactor is synthesized by UbiX.6,8 So far, the only prFMN-dependent enzyme for which the reaction has been biochemically characterized is ferulic acid decarboxylase (FDC).5,6,16−19 This enzyme decarboxylates a range of ring-substituted phenylacrylic acid (cinnamic acid) derivatives to produce the corresponding substituted styrene derivatives. FDC has attracted particular interest for its potential to produce a wide variety of commercially valuable substituted aromatic compounds such as styrene, vinyl guiacol and vanillin.2 The mechanism by which prFMN catalyzes decarboxylation remains unsettled. On the basis of crystal structures of FDC complexed with a variety of substrate analogs, it was proposed that decarboxylation involves an initial 1,3-dipolar cycloaddition between prFMN and the double bond of phenylacrylic acid to form a pentacyclic prFMN adduct.6 Decarboxylation then occurs by a Grob-type elimination of carbon dioxide in which the flavin nucleus acts as an electron sink. (Figure 2A). Protonation of this intermediate by an active site glutamate yields a styrene adduct, and finally the catalytic cycle is completed by cyclo-elimination to yield styrene and prFMN. This mechanistic proposal has attracted considerable interest because true thermal pericyclic reactions are extremely rare in enzymes and enzyme-catalyzed 1,3-dipolar cyclo-additions are unprecedented.20,21 So far, there is no direct experimental evidence to support the formation of a cyclo-addition adduct; in particular, no such adducts were observed among the various crystal structures solved for FDC.6 Moreover, whereas nitrogen ylides are well-known to undergo 1,3-dipolar cyclo-addition

E

nzyme-catalyzed decarboxylation reactions are an important class of biological reactions. They are also of interest as environmentally friendly catalysts that can be used to produce high value, optically pure chemicals under mild conditions.1−4 However, decarboxylation reactions on unactivated molecules are inherently difficult to achieve because of the buildup of negative charge on the α-carbon during the transition state. Nature has therefore evolved a wide variety of catalytic strategies to overcome this unfavorable energetic barrier; for example, employing organic prosthetic groups such as pyridoxal phosphate and thiamine pyrophosphate, and metals such as Mg2+, Fe2+ and Mn2+ that serve as Lewis acids to stabilize negative charge.1,2 The most recent decarboxylation cofactor to be discovered is a modified form of FMN in which the addition of an isopentyl group between the C6 and N5 positions of the isoalloxazine flavin nucleus results in the formation of a new 6-membered ring (Figure 1).5−7 This cofactor, which has been termed prenylated FMN (prFMN), is synthesized from reduced FMN and dimethylallyl phosphate by specialized prenyl transferases.8,9 It appears that prFMN-dependent enzymes (and their attendant prFMN synthases) are widely distributed in microbes where they are involved in the decarboxylation of various aromatic carboxylic acids.10 For example, in many prokaryotes ubiquinone biosynthesis involves the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid;11−15 in Escher© 2017 American Chemical Society

Received: May 16, 2017 Published: July 28, 2017 10972

DOI: 10.1021/jacs.7b05060 J. Am. Chem. Soc. 2017, 139, 10972−10975

Communication

Journal of the American Chemical Society

Figure 3. Scheme showing the possible reaction products of the substrate mimic, 2-fluoro-2-nitrovinylbenzene, with prFMN by either Michael addition or 1,3-dipolar cyclo-addition pathways.

reaction of FNVB with the enzyme could conveniently be followed by changes in the UV-absorption spectrum of FNVB (Figure 4A). The spectral changes showed an isosbestic point at 390 nm, indicative of a single chemical reaction, with the Figure 2. Alternative mechanistic proposals for the prFMN-dependent decarboxylation of phenylacrylic acid catalyzed by FDC. (A) An initial 1,3-dipolar cyclo-addition reaction facilitates decarboxylation of the substrate. (B) Michael addition of prFMN to the substrate is followed by elimination of CO2.

reactions,22 ylides are also intrinsically nucleophilic.23 Therefore, in the absence of any knowledge regarding the intrinsic chemical reactivity of prFMN, an alternative mechanism is also plausible that involves a Michael addition of prFMN to the double bond of phenylacrylic acid, followed by decarboxylation and elimination of the cofactor (Figure 2B). Previously, we examined the mechanism of FDC using series of substituted phenylacrylic acids to undertake a Hammett analysis of the reaction.18 Surprisingly, the Hammett ρ value for the reaction was negative (ρ = −0.38) indicating that decarboxylation was unlikely to be rate determining. This observation, together with deuterium kinetic isotope effect measurements, suggested that resolution of the productprFMN adduct was rate determining and further suggested an approach by which the putative cyclo-addition product might be stabilized. It is well established that cyclo-addition reactions of nitrogen ylides are dominated by the interaction of HOMO of the ylide and LUMO of the dipolarophile.24 Thus, lowering the energy of the dipolarophile LUMO results in a stronger interaction and a more stable cyclo-addition product. We reasoned that it might be possible to react FDC with a substrate analog with a lowenergy LUMO that would react with prFMN to form a sufficiently stable adduct to allow characterization (Figure 3). (Z)-2-Fluoro-2-nitro-vinylbenzene (FNVB) appeared to be a promising candidate, as the nitro-group is electron-withdrawing and an excellent isostere for a carboxylate group, whereas the fluorine is electronegative and a good isostere for hydrogen. Consistent with our prediction, FNVB proved to be a potent irreversible inhibitor of FDC. A 100 nM solution of FDC was reacted with 1 μM FNVB in 100 mM Na citrate buffer pH 6.5 at 25 C. After 10 min, assay of the enzyme with 50 μM phenylacrylic acid revealed FDC to be essentially inactive. The

Figure 4. (A) Spectral changes associated with the reaction of holoFDC with FNVB. Inset: time course for the reaction followed by the decrease in absorbance at 326 nm. (B) Difference spectra obtained after subtraction of holo-FDC spectrum: in black, initial absorbance due to FNVB; in red, and spectrum after reaction of holo-FDC with FNVB showing new absorbance band centered on 425 nm characteristic of a C4a,N5-dialkyl flavin. 10973

DOI: 10.1021/jacs.7b05060 J. Am. Chem. Soc. 2017, 139, 10972−10975

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Journal of the American Chemical Society greatest decrease in absorption occurring at 326 nm. This absorption band is associated with the extended π-system of FNVB and is replaced by a weaker, broad absorption band centered at 440 nm. The reaction was well described by a firstorder kinetic model with a rate constant of 0.64 ± 0.04 min−1. The reaction was independent of FNVB concentration, indicating the observed rate constant represents the true rate of reaction between FNVB and prFMN rather than FNVB binding to the enzyme. Flavins possess rich and informative electronic spectra25,26 that are indicative of both redox and protonation state and sensitive to changes in the substitution pattern of the isoalloxazine ring system.27 In particular, u.v.-visible spectra have been characterized for both C4a,N5-dialkyl flavins28 and N5-alkyl flavinium cations,29 which correspond to the reaction of FNVB with prFMN by either cyclo-addition or Michael addition, respectively (Figure 3). C4a,N5-dialkyl flavins have a characteristic absorbance at ∼400 nm that is sensitive to the electronic properties of other substituents on the flavin nucleus.28 In contrast, the N5-alkyl flavinium cation is characterized by absorption bands at 350 and 550 nm. The spectrum of the prFMN-FNVB adduct, with its broad absorbance band at 425 nm, (Figure 4A) is clearly indicative of the formation of a cyclo-addition adduct, rather than a Michael addition adduct. To provide further evidence to substantiate the cycloaddition mechanism, we next considered other spectroscopic techniques that could distinguish the two reaction pathways. In principle, the different reaction products could be distinguished by NMR and, in particular, the coupling patterns between the 19 F and 1H nuclei should be quite different between the two products. In practice, however, the small amount of material produced in the reaction and the instability of the adduct once dissociated from the protein precluded unambiguous identification of the reaction product by 19F or 1H NMR (data not shown). To overcome the problems associated with the instability of prFMN-FNVB adduct, we turned to native mass spectroscopy. This technique allowed us to introduce FNVB-inactivated holoFDC intact into the spectrometer, dissociate the cofactor from the protein and record its MS in situ, allowing the adduct no time to decompose. The cyclo-addition and Michael reaction products can readily be distinguished by MS because the Michael addition of prFMN to the double bond of FNVB involves protonation of the prFMN-FNVB adduct. This results in a positively charged adduct that is 1 amu heavier than the cyclo-addition adduct. (We note that the presence of fluorine raises the pKa of C1 of FNVB, estimated pKa ∼ 14,30 making it very unlikely that the Michael adduct would exist as the neutral zwitterion under the conditions of the experiment.) A solution of FDC (50 μM) in 100 mM Na citrate buffer, pH 6.5, that had been reacted with 50 μM FNVB for 10 min at room temperature was rapidly desalted through a P6̅ desalting column into 500 mM ammonium acetate buffer, pH 6.9. The inactivated holoenzyme was introduced into the mass spectrometer by electrospray ionization and the cofactor dissociated from the protein in situ by collision-induced dissociation under mild conditions (trap collision energy = 100 V). The spectrum of the cofactor (Figure 5) showed a prominent peak at m/z = 730.167 that exactly matches the mass expected for the cyclo-addition product of prFMN with FNVB complexed with one potassium ion (expected m/z = 730.169). This peak was not observed in control experiments in

Figure 5. Low m/z spectra of molecules released from FDC in situ by collision-induced dissociation; structure of the major molecular species shown on left. (A) FDC expressed and purified from E. coli lacking prFMN; the peak at 457.143 corresponds to a small amount of unmodified FMN. (B) holoFDC; the peak at 525.175 corresponds to prFMN. (C) FNVB-inactivated holoFDC; the peak at 730.167 corresponds to the K+ complex of the cyclo-addition adduct. The identities of the other peaks are discussed in the text.

which FNVB was omitted or when apo-FDC lacking prFMN was used (Figure 5). Peaks of lower m/z were also observed that represent the loss of HNO2 from the adduct (m/z = 683.189); the loss of HNO2 and exchange of K+ by H+ (m/z = 645.227); and small amount of unreacted prFMN (m/z = 525.175). As a further check that the m/z = 730.167 peak represents the cyclo-addition adduct, the isotope distribution of this peak was analyzed (Figure S1) and compared to the expected isotope distribution calculated from the natural abundance isotope distribution. The experimental and calculated distributions were found to be in excellent agreement. (The calculated isotope distribution for the Michael addition adduct was clearly incompatible with the spectrum.) The complex of FNBV-inactivated prFMN bound to FDC could also be observed at higher m/z prior to collision-induced dissociation (Figure S2). The mild ionization conditions associated with native MS preclude accurate molecular mass determination due nonspecifically bound solvent molecules. However, this observation serves as an additional control that the reaction of prFMN with FNVB occurs on the enzyme, rather than as a consequence of prFMN diffusing from the active site (the cofactor is lost upon dialysis)5 and reacting nonenzymatically with FNVB. These results provide the first direct experimental evidence that this novel and structurally complex flavin-derived cofactor is reactive toward 1,3-dipolar cyclo-addition chemistry. To our knowledge, this type of reaction has not previously been observed in an enzyme. This observation supports the proposed Grob-type decarboxylation mechanism6 in FDC that is facilitated by the formation of pentacyclic prFMNsubstrate adduct through cyclo-addition, and is consistent with our previous kinetic analyses of the enzyme.5,18 10974

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(13) Jacewicz, A.; Izumi, A.; Brunner, K.; Schnell, R.; Schneider, G. PLoS One 2013, 8, e63161. (14) Meganathan, R. FEMS Microbiol. Lett. 2001, 203, 131. (15) Rangarajan, E. S.; Li, Y. G.; Iannuzzi, P.; Tocilj, A.; Hung, L. W.; Matte, A.; Cygler, M. Protein Sci. 2004, 13, 3006. (16) Huang, Z. X.; Dostal, L.; Rosazza, J. P. N. J. Bacteriol. 1994, 176, 5912. (17) Mukai, N.; Masaki, K.; Fujii, T.; Kawamukai, M.; Iefuji, H. J. Biosci. Bioengin. 2010, 109, 564. (18) Ferguson, K. L.; Arunrattanamook, N.; Marsh, E. N. G. Biochemistry 2016, 55, 2857. (19) Lan, C. L.; Chen, S. L. J. Org. Chem. 2016, 81, 9289. (20) Pindur, U.; Schneider, G. H. Chem. Soc. Rev. 1994, 23, 409. (21) Baunach, M.; Hertweck, C. Angew. Chem., Int. Ed. 2015, 54, 12550. (22) Gothelf, K. V.; Jorgensen, K. A. Chem. Rev. 1998, 98, 863. (23) Jiang, K.; Chen, Y. C. Tetrahedron Lett. 2014, 55, 2049. (24) Pellissier, H. Tetrahedron 2007, 63, 3235. (25) Ghisla, S.; Massey, V.; Lhoste, J. M.; Mayhew, S. G. Biochemistry 1974, 13, 589. (26) Ghisla, S.; Massey, V. Eur. J. Biochem. 1989, 181, 1. (27) Ghisla, S.; Hartmann, U.; Hemmerich, P.; Muller, F. Annal. Der Chemie-Justus Liebig 1973, 1973, 1388. (28) Eckstein, J. W.; Hastings, J. W.; Ghisla, S. Biochemistry 1993, 32, 404. (29) Nanni, E. J.; Sawyer, D. T.; Ball, S. S.; Bruice, T. C. J. Am. Chem. Soc. 1981, 103, 2797. (30) Adolph, H. G.; Kamlet, M. J. J. Am. Chem. Soc. 1966, 88, 4761. (31) Leys, D.; Scrutton, N. S. Curr. Opin. Struct. Biol. 2016, 41, 19.

Homologues of FDC and PAD1 are found widely throughout microbial organisms. However, in most cases they appear to be involved in catalyzing decarboxylations on substrates in which the carboxylate group is directly attached to the aromatic ring, such as the decarboxylation of 4-hydroxy3-octaprenylbenzoic acid by UbiD.10,14,31 The decarboxylation of such substrates poses a much greater challenge for the cycloaddition mechanism because it implies that prFMN would have to react directly with the benzene ring. The resulting loss of aromatic character would appear to pose a significant energetic barrier to this type of reaction occurring. It will be interesting to see whether prFMN facilitates aromatic decarboxylation by a similar mechanism, or whether the decarboxylation of phenylacrylic acids by FDC is a mechanistic outlier.

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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/jacs.7b05060. Details of experimental methods; native MS spectra of FDC−FNVB−prFMN complexes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Brandon T. Ruotolo: 0000-0002-6084-2328 E. Neil G. Marsh: 0000-0003-1713-1683 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by grants from the National Science Foundation, CHE 1152055, CBET 1336636, to E.N.G.M.

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REFERENCES

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DOI: 10.1021/jacs.7b05060 J. Am. Chem. Soc. 2017, 139, 10972−10975