Letter Cite This: Org. Lett. 2018, 20, 1015−1018
pubs.acs.org/OrgLett
Enzymatic Synthesis of GDP-α‑L‑fucofuranose by MtdL and Hyg20 Xiangjing Qin,† Yunchang Xie,† Hongbo Huang,† Qi Chen,† Junying Ma,† Qinglian Li,† and Jianhua Ju*,†,‡ †
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China ‡ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 110039, China S Supporting Information *
ABSTRACT: Two mutases, MtdL and Hyg20, are reported. Both are able to functionally drive the biosynthesis of GDP-α-Lfucofuranose. Both enzymes catalyze similar functions, catalytically enabling the bidirectional reaction between GDP-β-L-fucopyranose and GDP-α-L-fucofuranose using only divalent cations as cofactors. This realization is but one of a number of important insights into fucofuranose biosynthesis presented herein.
F
ucose is a widespread natural sugar found in plants, animals, and microbes; its presence in natural products often enables a myriad of biological activities. For instance, fucose-containing glycans play important roles in nerve conduction, regulation of leukocyte−endothelial adhesion processes, human ABC-blood group antigens and related pathways, carcinogenesis pathways, and host−microbe interactions in humans and animals.1−3 Fucose is also a key factor in bacterial adhesion and growth4 and is the main component of plant cell walls as is the case for marine algae.5 Fucose possesses either the D- or L-stereochemical configuration; the L-configuration is the main stereoisomer found in nature. Both D-fucose and L-fucose contain six-membered pyranose and five-membered furanose forms, termed fucopyranose (Fucp) and fucofuranose (Fucf), respectively. However, Land D-fucofuranose are rare in nature due to their lower thermal stability relative to their Fucp congeners. Thus, far, D-Fucf is known to be a component of the O-antigen of Gram-negative bacteria such as Escherichia coli O52, Providencia rustigianii O34 and is a building block of the antitumor agents gilvocarcin V.6−8 L-Fucf is a crucial building block in some important naturally bioactive compounds, such as hygromycin A and 5′-dihydrohygromycin9 (antibacterial and immunosuppressant), fucoidan CF (anti-infective and antitumor),10−12 terpioside A,13 and terpioside B14 (anti-inflammatory and NO release inhibitors) (Figure 1). The catalytic generation of dTDP-D-Fucf from dTDP-Dfucopyranose (Fucp) by the mutase Fcf2 has been reported.15 Notably, divalent cations (Mg2+, Mn2+, Ca2+, Fe2+, Co2+) have no bearing on Fcf2 activity.15 In contrast, the enzyme responsible for generating the L-Fucf featured in natural products remains elusive. Recently, we reported a new pyranose−furanose mutase MtdL16 that interconverts GDP-β-L-galactopyranose (Galp) and GDP-α-L-galactofuranose (Galf) using divalent cations (Mn2+ or Mg2+) as cofactors. This system differs from that of the mutase UGM, which catalyzes the NDP-D-galactopyranose pyranose− furanose conversion using FAD as a cofactor.17,18 Thus, we © 2018 American Chemical Society
Figure 1. Structures of GDP-α-L-fucofuranose and natural products containing the α-L-fucofuranose moiety (red).
wondered if structurally related GDP-β-L-Fucp could be recognized as a substrate by MtdL and whether or not other homologous enzymes can catalyze the interconversion between GDP-β-L-Fucp and GDP-α-L-Fucf. Here we report (i) in vitro biochemical assays showing that both MtdL and Hyg20 can catalyze interconversion of GDP-β-LFucp and GDP-α-L-Fucf, (ii) that divalent cations such as Mn2+, Mg2+, Co2+ or Cu2+ are essential for the reaction, (iii) rigorous structural elucidation of the enzymatic product GDP-α-L-Fucf by HRMS and NMR data analysis, (iv) kinetics studies indicating that Hyg20 has greater substrate binding affinities than MtdL and that the reaction favors pyranose formation, and (v) that Hyg20 is also able to effect the interconversion of GDP-β-L-Galp and GDP-α-L-Galf. Received: December 20, 2017 Published: January 30, 2018 1015
DOI: 10.1021/acs.orglett.7b03962 Org. Lett. 2018, 20, 1015−1018
Letter
Organic Letters
Following this discovery with MtdL, we then assessed the activity of Hyg20 overexpressed using E. coli BL21 (DE3) as a soluble N-terminal 6 × His-tagged fusion protein (SI, Figure S2). Employing the same reaction and analysis conditions as had been applied to MtdL, we analyzed Hyg20s ability to catalyze the GDP-β-L-Fucp → GDP-α-L-Fucf conversion. As expected, a new peak with tR = 20 min was identified upon HPLC analyses of Hyg20 reactions (Figure 2, B). Functional Hyg20 was clearly required for new product generation, and the addition of divalent Mn2+ clearly enhanced the reaction whereas the addition of exogenous Mg2+ had little to no impact on reaction efficiency. Given that trace product could be detected in the absence of exogenous Mn2+ or Mg2+ suggests that some percentage of the overexpressed Hyg20 harbors divalent metal (Figure 2, B, trace v). This idea is supported by the fact that inclusion of EDTA abrogated product formation ( Figure 2, B, trace vi). In light of these data, it would appear that Mg2+ is a very poor cofactor for Hyg20 relative to Mn2+ and/or its biosynthetically incorporated cofactor. As had been the case with MtdL studies, further HRLCMS analyses revealed that the Hyg20 product mass ([M + H]+ m/z = 588.0691) was almost identical to that of the putative GDP-β-L-Fucp adduct ([M + H]+ m/z = 588.0698) (SI Figure S9). These data firmly suggest that Hyg20 and MtdL share similar, if not identical, enzymatic activities. For both enzymatic reactions ∼10% of GDP-β-L-Fucp was converted into new product (tR = 20 min) and final equilibrium ratios (on the basis of HPLC peak integrations) for GDP-β-L-Fucp: GDP-α-L-Fucf under the artificial reaction conditions employed were ∼9:1. Time course studies with Hyg20 and GDP-β-L-Fucp as substrate revealed that the conversion of GDP-β-L-Fucp to GDPα-L-Fucf was time dependent, proceeding to 8% conversion after 10 min (Figure 3). The reaction approached its equilibrium point
Bioinformatics analysis revealed that Hyg20 shares 59% sequence homology with MtdL [Supporting Information (SI), Figure S1]. From this was inferred that Hyg20 is likely responsible for the conversion of NDP-L-Fucp to NDP-L-Fucf, in the biosynthesis of the aminoglycoside antibiotic hygromycin; however, this was not experimentally verified.19,20 Both Hyg20 and MtdL contain the conserved DxD motif, important to NDPsugar binding and the enzymatic activity of many microbial and mammalian type-A glycosyltransferases (GT-A).21,22 These GTAs catalyze the formation of glycosidic bonds and require divalent cations (Mn2+ or Mg2+) as essential cofactors and nucleotide diphosphate (NDP) sugars as substrates. Structurally, GDP-β-L-Fucp is 6-deoxy GDP-β-L-Galp. We first assessed the ability of MtdL to catalyze the GDP-β-LFucp → GDP-α-L-Fucf transformation. We have previously reported the expression and purification of MtdL16 and the substrate GDP-β-L-Fucp is commercially available. GDP-β-LFucp (1 mM) was incubated with 5 mM Mg2+ or Mn2+ in 50 μL of 50 mM PBS (pH 8.0) and 2 μM MtdL at 37 °C. The reaction was initiated by addition of MtdL and, following incubation for 30 min, was terminated by heating to 100 °C for 2 min. The reaction mixture was centrifuged for 15 min at 14000 rpm and the supernatant then analyzed by HPLC using a mobile phase of 50 mM triethylammonium acetate (TEAA, pH 6.5) containing 1.5% acetonitrile and detection at λ = 254 nm detector; a flow rate of 0.7 mL/min was employed. Under these conditions, the tR of GDP-β-L-Fucp was found to be 15 min, and a new product at tR = 20 min (Figure 2, A, traces iii and iv) became evident. The same
Figure 2. MtdL (A) and Hyg20 (B) catalyze formation of GDP-α-L-Fucf using GDP-β-L-Fucp as substrate: (i) GDP-β-L-Fucp standard, (ii) reaction with heat denatured MtdL or Hyg20, (iii) reaction with Mg2+, (iv) reaction with Mn2+, (v) reaction without exogenously added Mg2+ or Mn2+, (vi) reaction in the presence of metal chelating EDTA. All reactions were carried out at 37 °C for 30 min. ●: GDP-β -L-Fucp. ▽: GDP-α-L-Fucf.
Figure 3. Time course of Hyg20-catalyzed reaction using GDP-β-L-Fucp as substrate. HPLC analyses of the in vitro biochemical reaction mixture at 2 min (ii), 5 min (iii), 10 min (iv), 15 min (v), 20 min (vi), and 60 min (vii), showing the Hyg20-catalyzed interconversion between GDP-β-LFucp and GDP-α-L-Fucf reached equilibrium at 20 min. Trace i was a negative control with heat-denatured Hyg20.
reaction, carried out using heat-inactivated MtdL, failed to afford this new product, suggesting the requirement of active MtdL (Figure 2, A, trace ii). The reaction also was found to require a divalent cation since the inclusion of 5 mM EDTA (Figure 2, A, trace vi) abrogated new product formation as did the absence of exogenously added metal. Further HR−LCMS analysis indicated that the new enzymatic product ([M + H]+ m/z = 588.0715) had a mass almost identical to that of the substrate GDP-β-L-Fucp ([M + H]+ m/z = 588.0710) (SI, Figure S8). Consequently, we hypothesized that the new MtdL-dependent adduct was the putative GDP-α-L-Fucf.
at a reaction time of 20 min, and Keq was determined to be 0.13. These in vitro biochemical results demonstrate that Hyg20 indeed catalyzes GDP-β-L-Fucp conversion to GDP-α-L-Fucf in a time-dependent manner. To structurally elucidate the product (tR = 20 min) of Hyg20/ MtdL catalysis, we scaled up enzymatic production of putative produce GDP-α-L-Fucf. The enzyme reaction was carried out in a volume of 500 μL consisting of 1 mg GDP-β-L-Fucp, 5 mM 1016
DOI: 10.1021/acs.orglett.7b03962 Org. Lett. 2018, 20, 1015−1018
Letter
Organic Letters Mn2+, and 50 μM MtdL or Hyg20 in 50 mM PBS (pH 8.0). The enzyme was removed by centrifugal filtration after incubation at 37 °C for 3 h and both substrates and products purified by HPLC. Since the conversions catalyzed by Hyg20 and MtdL evidently favor formation of GDP-β-L-Fucp, we repeated the above steps until amounts of putative product sufficient to support structure elucidation efforts could be obtained. Finally, the purified product was lyophilized and dissolved (0.6 mg) into 500 μL PBS in D2O (pH 8.0). 1H and 1H−1H TOCSY NMR spectra (SI, Figures S10−13) were then acquired enabling us to make the following 1H NMR assignments: guanine unit (G): δ 8.07 (1H, s, H-G8); ribose unit (R): δ 5.90 (1H, d, J = 6.3 Hz, HR1), 4.75 (overlapped, H-R2), 4.49 (lH, J = 5.1, 3.8 Hz, H-R3), 4.32 (1H, m, H-R4), 4.17 (2H, m, H-R5); α-L-fucofuranose unit (F): δ 5.56 (lH, d, J = 5.9 Hz, H−F1), 4.19 (lH, m, H−F2), 3.87 (lH, dd, J = 5.3, 2.4 Hz, H−F3), 3.94 (1H, dd, J = 6.5, 5.3 Hz, H− F4), 3.82 (lH, m, H−F5), 1.19 (3H, d, J = 6.5 Hz, H−F6). The coupling constant of the anomeric proton signal (JH1−H2 = 5.9 Hz), confirmed that the product is, indeed, GDP-α-L-Fucf.23,24 In addition, we tested the stability of GDP-α-L-Fucf; we observed no spectroscopic changes following 24 h at either 4 or 25 °C (SI, Figure S3). These in vitro data further validate that Hyg20 and MtdL catalyze conversion of GDP-β-L-Fucp into GDP-α-L-Fucf. We next investigated the reversibility of MtdL- and Hyg20mediated chemistry. The ability of both enzymes to convert GDP-α-L-Fucf to GDP-β-L-Fucp employed reaction conditions identical to those used previously barring the obvious substrate change. As anticipated, HPLC analyses of this reaction (Figure 4)
cofactor than Mg2+; this was especially pronounced for MtdL where the impact of exogenously added Mg2+ was almost negligible (Figure 4, A, traces v−vii). These data (Figure 4) also revealed that the enzymatic conversion of GDP-α-L-Fucf to GDP-β-L-Fucp is significantly more facile than the reverse transformation. The catalytic rate of Hyg20 was found to be much greater than that of MtdL on the basis of HPLC peak integrations. The GDP-α-L-Fucf → GDP-β-L-Fucp conversion ultimately reached equilibrium with a ratio for GDP-β-LFucp:GDP-α-L-Fucf of 9:1. All these data indicate that Hyg20 and MtdL do, indeed, catalyze the GDP-β-L-Fucp ⇆ GDP-α-LFucf interconversion and that Hyg20 is a much more active mediator of this chemistry than is MtdL. To better understand this unique transformation systematic investigation of the effects of divalent cations, pH, and temperature on MtdL- and Hyg20-mediated chemistry was carried out. In light of the differences seen with Mg2+ and Mn2+ in influencing the enzymatic GDP-α-L-Fucf ⇆ GDP-β-L-Fucp interconversion, we examined more closely the effects of these metals as well as Ca2+, Cu2+, Co2+, Fe2+, and Ba2+ on the activity of Hyg20 (SI, Figure S4, A). Trials with [Mn2+] = 1 mM versus 10 mM (SI, Figure S4, B) revealed little difference in activity and trials with [Mn2+] at 5 mM revealed little that Mn2+, Mg2+, Fe2+, Co2+, and Cu2+ stimulated furanose generation by Hyg20; all other cations had no apparent impact on Hyg20 activity. Notably, the use of Fe2+ led to a number of otherwise minor/ absent byproducts including GDP ([M + H]+ m/z = 440.00) and GMP ([M + H]+ m/z = 361.04). We also noted that Cu2+ and Co2+ impacted Hyg20 activity in a fashion highly similar to that observed with Mn2+. Not surprisingly, all metal-dependent reactions were completely abrogated by 5 mM EDTA. On the basis of these findings, it is evident that both Hyg20 and MtdL require the presence of divalent cations. These divalent cations may play a key role in stabilizing substrates, intermediates, or the relevant products and key residues of the MtdL/Hyg20 active site regions such as the DxD motif. Hyg20-mediated conversion rates were determined at different temperatures ranging from 4 to 75 °C (SI, Figure S5). Interestingly, Hyg20 was temperature sensitive; the enzyme effectively catalyzed formation of GDP-αL-Fucf even at 45 °C. The Hyg20-catalyzed reaction was found to achieve equilibrium at 37−45 °C, although the conversion ratio was low, being less than 2% at 4−25 °C. The enzyme showed no catalytic activity at temperatures above 60 °C. The influence of pH on Hyg20 and MtdL activity was also investigated; the optimal pH for GDP-β-L-Fucp ⇆ GDP-α-L-Fucf interconversion was found to be 7.6−8.0 (in 50 mM PBS) (SI, Figure S6). At this pH range no byproducts were evident; at pH = 5.8−6.8, at least three byproducts were observed with GMP being especially pronounced at pH = 5.8. Using the optimal reaction conditions described above, we determined the kinetic constants of MtdL and Hyg20 with 5 mM Mn2+. The Km and kcat for MtdL in converting GDP-α-L-Fucp to GDP-α-L-Fucf were found to be 605.2 μM and 4.31 min−1 respectively, and for the reverse reaction were 73.53 μM and 6.85 min−1 (Figure 5, A and C). Thus, the rate for the reverse MtdL reaction is higher than that of the forward “furanose generating” reaction. This is likely due to the higher affinity of MtdL for GDP-α-L-Fucf than its pyranose congener, as well as the greater efficiency of MtdL in producing the pyranose adduct rather than the furanose species. The Km and kcat of Hyg20 for GDP-α-LFucp were only 1/5 and 1/3 those of MtdL (Figure 5, A and B), respectively, indicating that Hyg20 is more easily saturated by substrate than is MtdL, and therefore has greater catalytic
Figure 4. MtdL (A) and Hyg20 (B) catalyze formation of GDP-β-LFucp using GDP-α-L-Fucf as substrate: (i) GDP-α-L-Fucf control, (ii) GDP-β-L-Fucp control, (iii) reaction with boiled MtdL/Hyg20, (iv) reaction with Mn2+, (v) reaction with Mg2+, (vi) reaction without Mg2+/ Mn2+, (vii) reaction with EDTA. All reactions were carried out at 37 °C for 30 min. ●: GDP-β-L-Fucp. ▽: GDP-α-L-Fucf.
revealed that both enzymes generated GDP-β-L-Fucp (as verified by HR-LCMS and HPLC comparisons to a standard). As had been the case previously, the pyranose adduct eluted with tR = 15 min and the furanose substrate eluted with tR = 20 min. Again too, divalent cations (Figure 4, traces iii and iv) appeared to play a key role in the reverse reaction although MtdL again displayed a low level of activity in the absence of exogenous metal consistent with low levels of metal in our overexpressed MtdL. This was supported by the realization that EDTA very effectively shut down both MtdL and Hyg20 (Figure 4, traces vii). As had been noted for the forward reaction, Mn2+ proved a more effective 1017
DOI: 10.1021/acs.orglett.7b03962 Org. Lett. 2018, 20, 1015−1018
Organic Letters
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hongbo Huang: 0000-0002-5235-739X Jianhua Ju: 0000-0001-7712-8027 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (81425022, U1501223, U1706206, and 31500057) and the Natural Science Foundation of Guangdong Province (2017A030313140).
Figure 5. Kinetic constants of MtdL- and Hyg20-catalyzed reactions. A (MtdL) and B (Hyg20) represent the reaction using GDP-α-L-Fucp as substrate. C (MtdL) and D (Hyg20) show the reverse reaction using GDP-α-L-Fucf as substrate. The inset numbers represent the determined kinetic constant values. Error bars are exhibited as SEM.
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efficiency than MtdL. However, the kcat value of Hyg20 (kcat = 3.66 min−1) in the reverse reaction using GDP-α-L-Fucf as a substrate was comparable to that of MtdL (kcat = 6.85 min−1), and the Hyg20 Km (6.671 μM) was only 1/11 those of MtdL (73.53 μM) (Figure 5, C and D) suggesting that the affinity of Hyg20 was 11-fold greater than MtdL. All these data indicate that the rate of Hyg20-catalyzed reaction is much higher than that of the MtdL-catalyzed reaction. Moreover, although Hyg20 is able to use GDP-β-L-Fucp as a substrate as reflected in Figures 2 and 3, GDP-α-L-Fucf is its preferred substrate. Finally, we also tested the ability of Hyg20 to employ GDP-βL-Galp as a substrate in generating its furanose counterpart. HPLC analyses indicated that, as with the previously studied MtdL system,16 GDP-β-L-Galp, in the presence of Hyg20 and Mn2+, was enzymatically converted to GDP-α-L-Galf (SI, Figure S7). This reaction was remarkably different from that of the DGalp and D-Glaf interconversion which is catalyzed by UGM and its FAD cofactor.17,18 The efficiency of the Hyg20-mediated process appeared to be on par with that of a parallel MtdLcontaining reaction. Hence, both MtdL and Hyg20 appear to catalyze pyranose−furanose interconversions with some degree of substrate flexibility. In summary, our in vitro assays reveal that both MtdL and Hyg20 catalyze GDP-β-L-Fucp ⇆ GDP-α-L-Fucf interconversion albeit with differing efficiencies. The reactions rely, to differing extents, on divalent cations (Mn2+, Mg2+, Co2+ or Cu2+) and thermodynamically favors the pyranose form, with a ratio of 9:1 (pyranose/furanose) at equilibrium. The enzymatic product GDP-α-L-Fucf was successfully isolated and elucidated by MS and NMR data analysis; the purified GDP-α-L-Fucf was found to be quite stable. This work expands our knowledge of this family of mutases and provides a mild means of preparing GDP-α-LFucf which is otherwise not readily achievable via conventional organic synthesis.
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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.orglett.7b03962. General experimental procedures, in vitro enzymatic assays, and NMR data and spectra for enzymatic products (PDF) 1018
DOI: 10.1021/acs.orglett.7b03962 Org. Lett. 2018, 20, 1015−1018
