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pH-rate profiles of l -arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in l -xylulose production
Bioorganic & Medicinal Chemistry Letters 24 (2014) 173–176
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
pH-rate profiles of L-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in L-xylulose production Manish Kumar Tiwari a,b,c, , Raushan Kumar Singh a, Hui Gao a, Taesu Kim a, Suhwan Chang d, Han S. Kim b,c,⇑, Jung-Kul Lee a,b,⇑ a
Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea Institute of SK-KU Biomaterials, Konkuk University, Seoul 143-701, Republic of Korea Department of Environmental Engineering, Konkuk University, Seoul 143-701, Republic of Korea d Department of Biomedical Sciences, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 June 2013 Revised 8 November 2013 Accepted 20 November 2013 Available online 27 November 2013 Keywords: Rare sugar L-Xylulose pH-Dependent kinetics Molecular dynamics
a b s t r a c t L-Arabinitol
4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of L-arabinitol oxidation by NAD+, catalyzed by HjLAD, was studied within the pH range of 7.0–9.5 at 25 °C. The turnover number (kcat) and the catalytic efficiency (kcat/ Km) were 4200 min 1 and 290 mM 1 min 1, respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare L-sugar L-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%. Ó 2013 Elsevier Ltd. All rights reserved.
L-Xylulose (threo-pentulose) is a ketopentose that is rare and expensive.1 In recent years, interest in L-carbohydrates and the L-nucleosides derived from them has substantially increased in medicine.2 Recent investigations have revealed that rare carbohydrates are potentially important for use as low-calorie carbohydrate sweeteners and bulking agents3,4 as well as potential inhibitors of various glycosidases.5,6 Moreover, the use of derivatives of an L-rare sugar as antihepatitis B virus agents has also been reported.7 Rare L-forms of sugars, such as L-xylose, have been used as starting materials for the synthesis of various pharmaceutically interesting molecules,8 including L-ribose, D-psicose, L-xylose, D-tagatose, and L-xylulose. Nucleoside analogues of L-sugars have potential as anticancer and cardioprotective agents, and they are being exploited as antiviral drugs against hepatitis-B and HIV.5,6 Recent reports suggest that novel L-xylose derivatives act as inhibitors of urinary glucose reabsorption, suggesting their application in diabetes treatment.9 Advancements in genomics, screening, and evolution technologies may lead to an increased availability of new and robust biocatalysts for industrial-scale application,
⇑ Corresponding authors. Tel.: +82 2 450 3505; fax: +82 2 458 3504 (H.S.K. and J.-K.L.). E-mail addresses: [email protected] (H.S. Kim), [email protected] (J.-K. Lee). Present address: Department of Chemistry, Technical University of Denmark, Kongens Lyngby 2800, Denmark. 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.047
and these advancements are stimulated by an increasing demand for catalysts that are used as ingredients.10 Biocatalysis often offers advantages over chemical synthesis, because enzyme-catalyzed reactions are often highly enantioselective and regioselective.10,11 These reactions can be conducted at an ambient temperature and atmospheric pressure, thereby avoiding the use of extreme conditions that can cause problems with isomerization, racemization, epimerization, and rearrangement.12 Therefore, enzymatic isomerization of the ketosugar L-xylulose to L-xylose is considered an alternative for chemical synthesis.13–15 The starting material L-xylulose can be produced by oxidation of a polyol substrate such as L-arabinitol by using recombinant catalysts.16–18 An efficient production method for L-xylulose would allow utilization of this rare sugar in new applications of commercial value.19L-Arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12), a common enzyme found in yeasts and filamentous fungi, catalyzes the second step of the fungal L-arabinose metabolic pathway by oxidizing L-arabinitol to
Figure 1. Bioconversion of L-arabinitol into L-xylulose using LAD.
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(Fig. 1) with concomitant NAD+ reduction.16,20 Both are intermediates in catabolic pathways for pentose and pentitol utilization in many organisms.19 This enzyme is known to operate by an effectively ordered ternary complex mechanism, in which coenzyme-binding precedes substrate binding.21 The stoichiometry of the overall reaction prescribes that a proton is liberated for every NADH molecule formed, indicating that the catalytic reaction is critically dependent on one or several proton transfer steps. Such proton transfers are expected to involve ionizing groups at the active center of the enzyme.22 Few studies have reported the kinetic parameters of LADs, and the reported values were largely dependent on experimental conditions such as the pH and buffer used. However, pH dependence of LAD has not been thoroughly investigated to date, and important aspects of pH-dependence and its relationship with kinetic behavior of LAD remain unclear. In addition, to obtain rare sugars on an industrial scale, a less-expensive production method with high yield is required. Therefore, it is highly desirable to identify conditions for LAD enzyme that can exhibit high catalytic efficiency. In the present Letter, we have reported pH-dependent kinetics of fungal LAD from Hypocrea jecorina (HjLAD) and its application in the production of L-xylulose. In an attempt to investigate the pH-dependent kinetics of HjLAD and the production of L-xylulose, the HjLAD gene was cloned into the T7 promoter-based plasmid pET28a to form pET28a-HjLAD, and it was then heterologously expressed23 in Escherichia coli BL21 (DE3) (see Supplementary data). The enzyme was purified using Ni-nitrilotriacetic acid affinity chromatography (Fig. 2), and the purified HjLAD showed high activity toward L-arabinitol oxidation. Using gel filtration chromatography on the Sephacryl S-300 High Resolution media (column 16/60; Amersham, UK), we found that HjLAD corresponded to a molecular weight of approximately 174 kDa (Fig. S1). The subunit molecular weight of the enzyme was approximately 41 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 2). These results suggest that HjLAD is a noncovalently linked tetramer in its native form. HjLAD showed activity in the pH range of 7.0–10.5, with the L-xylulose
L-and L-xyluloses
Figure 2. Overexpression and purification of recombinant HjLAD. Lane M, the molecular weight marker proteins (kDa). Lane 1, whole-cell fraction of recombinant HjLAD. Lane 2, insoluble protein fraction. Lane 3, 10 lg of soluble protein fraction of recombinant BL21 (DE3/pET28a-HjLAD). Lane 4, 4 lg of purified HjLAD enzyme.
maximum activity observed at pH 9.5 (Fig. S2). The enzyme activity was significantly reduced above pH 9.5 and below pH 7.0, and approximately 10% and 40% of the enzyme activity was retained at pH 7.0 and 10.5, respectively (Fig. S2). However, NAD+ appeared to be unstable at pH values above 10.5 during extended incubation.19,24 Therefore, enzymatic activities in this study were routinely determined in the pH range of 7.0–9.5. The optimal temperature for oxidation of L-arabinitol was 65 °C (Fig. S3). Under optimum assay conditions, the kinetics of L-arabinitol oxidation, catalyzed by LAD, were studied in the pH range of 7.0–9.5 at 25 °C. The reactions were conducted in 100 mM Tris–HCl (pH 7.0–8.0) and Tris–glycine–NaOH (pH 8.5–9.5). Initial rates were determined by measuring changes in absorbance at 340 nm by using an ultraviolet (UV)–visible spectrophotomer at room temperature.24 Various concentrations of the substrate (L-arabinitol; 25– 500 mM) and cofactors (NAD+; 0.25–2 mM) were used to determine the kinetic parameters. Enzyme kinetics for the substrate and cofactors were analyzed using Michaelis–Menten kinetics, and the kinetic parameters were determined by fitting data to the non-linear regression plot.24 The catalytic activity of HjLAD is strongly dependent on pH over the pH range of 7.0–9.5 (Table 1). Maximal HjLAD activity at an alkaline pH for alcohol dehydrogenation of sugar is a common feature of similar enzymes isolated from diverse microbial systems.24,25 Specific activity of HjLAD was reported to be between 0.013 and 1.6 U/mg;16,26 these values were obtained with partially purified protein or a subsaturated cofactor concentration (0.25 mM) with purified enzyme. The specific activity of Trichoderma longibrachiatum LAD reported by Kim et al. using recombinant His-tagged purified protein was 8.7 U/mg at pH 7.0.27 In this study, at pH 7.0, Km of HjLAD was 18.2 ± 1.2 mM for L-arabinitol and 0.20 ± 0.03 mM for NAD+; these values were consistent with those reported in the literature.16,26,27 However, at pH 9.5, the specific activity of HjLAD was 105 ± 9 U/mg-protein, which is significantly higher than that previously reported, and Km of HjLAD was 14.5 ± 1.0 mM for L-arabinitol, which is slightly lower than that reported in previous studies, thereby resulting in much higher catalytic efficiency (kcat/Km = 290; Table 1). It is clear from the results in Table 1 and Fig. 3 that the catalytic rate of HjLAD was essentially dependent on the pH range 7.0–9.5. At pH 7.0, the kcat value of HjLAD enzyme decreased to approximately 22% of that at pH 9.5. Table 2 shows a comparison of the properties of various LADs from different sources. It is noteworthy that HjLAD shows the highest turnover rate (kcat) and catalytic efficiency (kcat/Km) among all previously characterized LADs. The kcat and kcat/Km of HjLAD were 1760 min 1 and 102 mM 1 min 1, and 4200 min 1 and 290 mM 1 min 1 at pH 8.0 and 9.5, respectively. It has been established that this pH dependence is derived from the combined effects of pH on several reaction steps in a catalytic mechanism; proton equilibria involving ionizing groups on the enzyme are known to affect the binding of both coenzyme and substrate, as well as the inter-conversion of the productive ternary complexes.29 Based on the study on pH-dependence of the reactions of yeast alcohol dehydrogenase, Dickenson et al. proposed
Table 1 Kinetic properties of HjLAD enzymes on various pH range 7.0 to 9.5 pH
Specific activity (U/mg)
9.5 9.0 8.6 8.0 7.5 7.0
105 ± 9 99 ± 5 96 ± 7 44 ± 2 36 ± 2 24 ± 1
Km (mM)
kcat/Km, (mM
Coenzyme
Substrate
0.50 ± 0.08 0.40 ± 0.08 0.40 ± 0.05 0.50 ± 0.02 0.20 ± 0.07 0.20 ± 0.08
14.5 ± 1.0 14.2 ± 1.0 17.8 ± 1.3 17.2 ± 1.6 16.4 ± 2.3 18.2 ± 1.2
289 ± 32 278 ± 30 215 ± 25 102 ± 13 87.8 ± 17.4 52.7 ± 10.1
1
min
1
)
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Figure 3. pH dependence of the turnover of enzymatic L-arabinitol oxidation.
that the transition state for alcohol oxidation is favored by the presence of a basic group in the vicinity of the hydroxyl group of the alcohol substrate. This basic group becomes protonated when hydride transfer occurs.30 However, the uptake of the proton in the course of the forward reaction can occur directly from the solvent or, alternatively, a catalytic residue at the enzyme active site can function in acid–base catalysis.31 Studies on steady-state kinetics of sheep live sorbitol dehydrogenase suggest that the kinetic constants vary with pH in accordance with a single pK of 7.1 for sorbitol oxidation.32 Furthermore, the pK value was assigned to the ionization properties of a catalytically essential group, and the pK values found for sorbitol oxidation referred to a histidine residue.33 In the present study, to depict the microenvironment of HjLAD active site, we derived a computational structure of HjLAD and docked with L-arabinitol.21,34 This structure was modeled based on a recently reported crystal structure of LAD from Neurospora crassa22 (NcLAD; Protein Data Bank accession No.: 3M6I), which shares 80% of the sequence identity with that of HjLAD.34 HjLAD showed a similar residual positioning; the Cys66, His91, Glu92, and Glu176 residues were involved in coordination of catalytic Zn2+ along with a water molecule (Fig. 4). Sequence alignment between HjLAD and other homologous protein sequences coupled with molecular dynamics suggests that the His91 is conserved in the more distant members of the alcohol/sorbitol dehydrogenase family. His91 in HjLAD (Fig. 4) is present in SDHs, including the liver enzyme from rat, sheep, and humans,35 as well as in Bacillus subtilis36 and yeast SDH,37
Figure 4. Homology modeling and docking of L-arabinitol in the substrate-binding pocket of HjLAD suggesting routes of hydride transfer, where the key amino acid residues comprise the core region. Carbon atoms of active-site residues and nicotinamide ring are in white; carbon atoms in L-arabinitol are in green. Nitrogen, oxygen, and sulfur atoms are in blue, red, and yellow, respectively.
and it is equivalent to His68 in sheep liver alcohol dehydrogenase38 and His70 in human sorbitol dehydrogenase.39 On the basis of the identity of the catalytic microenvironment at the active site of HjLAD and related enzymes, one may hope to observe similarities in the reaction at the catalytic center. Furthermore, the indispensable role of His91 in catalysis was confirmed by site-directed mutagenesis. H91A mutant enzyme was successfully overexpressed with bands displaying a molecular mass of approximately 40 kDa, as expected for a monomer of LAD (Fig. S4A). The secondary structure of the H91A was analyzed along with the wild-type HjLAD.34 The wild-type enzyme and the purified protein from H91A mutant exhibited similar circular dichroism (CD) spectra, with ellipticity minima of comparable amplitude in the range of 220–240 nm. The CD spectra of all of the native proteins were very similar (Fig. S4B). This indicated that all tested wild-type and mutant enzymes had a similar secondary structure and that the global folding of mutant and wild-type HjLAD is similar. However, despite its efficient expression and similar secondary structure, the HjLAD mutant (H91A) was inactive, which demonstrates its crucial role in catalysis. Using thermostable27 and highly active biocatalyst HjLAD, L-xylulose production was carried out at pH 9.5 and 7.0 (see Supplementary data). In the present study, L-arabinitol, a naturally occurring polyol that is commercially available, was used as the
Table 2 Biochemical and kinetic properties of LAD enzymes from various organisms Organism
Hypocrea jecorina Aspergillu oryzae Neurospora crassa Hypocrea jecorina Aspergillus niger Trichoderma longibrachiatum Penicillium chrysogenum Aspergillus niger Hypocrea jecorina Hypocrea jecorina Hypocrea jecorina NR: not reported.
Optimum pH
Kinetics pH
NR NR 9.5 NR 9.4 9.4
7.0 NR 8.0 7.0 7.0 7.0
9.4 NR 9.5 9.5 9.5
7.0 9.6 7.0 8.0 9.5
Specific activity (U/mg) 1.6 0.04 31 0.01 11.7 8.7 25.3 96 24 44 105
Km (mM)
kcat (min
1
)
kcat/Km, Larabinitol (mM 1 min
References
Coenzyme (NAD+)
Substrate (L-arabinitol)
0.18 NR 0.17 NR 0.2 0.2
40 NR 16 4.5 25 18
NR NR 1206 51 507 346
NR NR 75 11 20 19
16 17 18 26 27 27
0.3 0.5 0.2 0.5 0.2
37 89 18.2 17.2 14.5
1085 NR 960 1760 4200
29 NR 53 102 290
27 28 This study This study This study
1
)
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(Grant 2011-50210). This work was also supported by the NRF Grant funded by the Korea government (2012M3C5A1053339). This work was supported by a Grant from the Next-Generation BioGreen 21 program (No. PJ009605), Rural Development Administration, Republic of Korea. This research was also supported by the 2012 KU Brain Pool of Konkuk University. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 11.047. References and notes
Figure 5. Time course production of L-xylulose using recombinant HjLAD at pH 9.5 (j) and pH 7.0 (N). The error bars show the standard deviation for three determinations.
starting material. The time courses of L-xylulose production at pH 9.5 and 7.0 are presented in Fig. 5. In the reaction mixture initially containing 5 g l 1 L-arabinitol, the conversion of L-arabinitol to Lxylulose reached 86 ± 3% and a volumetric productivity of 2.2 g l 1 h 1 was obtained after 2 h of reaction at pH 9.5. While at pH 7.0, L-xylulose productivity was significantly decreased. After 2 h of reaction, only 42 ± 4% conversion was obtained. In the control experiment without LAD enzyme, no L-arabinitol consumption or L-xylulose production was detected during 4 h of incubation of the reaction mixture. The current production study is favorably comparable with L-xylulose production rates reported in the literature.19,40 The highest L-xylulose yields from xylitol of 70% for Alcaligenes sp. strain 701B41 and 80% for Pantoea ananatis ATCC 4307219 were obtained at a xylitol concentration of 5 g l 1 in 24 and 18 h, respectively. A yield of approximately 86% was achieved within 2 h with the recombinant purified HjLAD in the present study. We further purified L-xylulose using ion-exchange column chromatography (see Supplementary data). As a result, we were able to obtain 99% pure L-xylulose (3.9 g) from L-arabinitol (5 g) in 78% yield. The purified L-xylulose was characterized by high resolution electrospray ionization mass spectrometry (HR-ESI MS) and 1 H NMR spectroscopy (see Supplementary data text, Figs. S5 and S6). The HR-ESI-MS (negative-ion mode [M–H] ) revealed a molecular weight of 149.0280 corresponding to the molecular formula of L-xylulose, C5H10O5. In summary, the gene (HjLAD) encoding LAD was cloned from H. jecorina and overexpressed in a soluble form. The purified HjLAD protein exhibited the highest LAD activity. By analogy with other reductases/dehydrogenases that share similarity at the sequence level, we speculated that the conserved His91, a potential activesite acid residue, could function in acid–base catalysis. To the best of our knowledge, this is the first study demonstrating the feasibility of LAD (HjLAD) in the production of rare sugars such as L-xylulose, which gave a yield of approximately 86% in 2 h. Therefore, HjLAD can be effectively utilized in biotechnological production of this value-added rare sugar. Acknowledgments This research was supported by the Converging Research Center Program through the National Research Foundation of Korea
1. Usvalampi, A.; Kiviharju, K.; Leisola, M.; Nyyssola, A. J. Ind. Microbiol. Biotechnol. 2009, 36, 1323. 2. Beerens, K.; Desmet, T.; Soetaert, W. J. Ind. Microbiol. Biotechnol. 2012, 39, 823. 3. Muniruzzaman, S.; Pan, Y. T.; Zeng, Y.; Atkins, B.; Izumori, K.; Elbein, A. D. Glycobiology 1996, 6, 795. 4. Livesey, G.; Brown, J. C. J. Nutr. 1996, 126, 1601. 5. Gumina, G.; Chong, Y.; Choo, H.; Song, G. Y.; Chu, C. K. Curr. Top. Med. Chem. 2002, 2, 1065. 6. Mathe, C.; Gosselin, G. Antiviral Res. 2006, 71, 276. 7. Moses, V.; Ferrier, R. J. Biochem. J. 1962, 83, 8. 8. Fishman, P.; Bar-Yehuda, S.; Madi, L.; Cohn, I. Anticancer Drugs 2002, 13, 437. 9. Goodwin, N. C.; Mabon, R.; Harrison, B. A.; Shadoan, M. K.; Almstead, Z. Y.; Xie, Y.; Healy, J.; Buhring, L. M.; DaCosta, C. M.; Bardenhagen, J.; Mseeh, F.; Liu, Q.; Nouraldeen, A.; Wilson, A. G.; Kimball, S. D.; Powell, D. R.; Rawlins, D. B. J. Med. Chem. 2009, 52, 6201. 10. Panke, S.; Wubbolts, M. Curr. Opin. Chem. Biol. 2005, 9, 188. 11. Pollard, D. J.; Woodley, J. M. Trends Biotechnol. 2007, 25, 66. 12. Patel, R. N. Coord. Chem. Rev. 2008, 252, 659. 13. Usvalampi, A.; Turunen, O.; Valjakka, J.; Pastinen, O.; Leisola, M.; Nyyssola, A. Enzyme Microb. Technol. 2011, 50, 71. 14. Bhuiyan, S. H.; Ahmed, Z.; Utamura, M.; Izumori, K. J. Ferment. Bioeng. 1998, 86, 513. 15. Granstrom, T. B.; Takata, G.; Morimoto, K.; Leisola, M.; Izumori, K. Enzyme Microb. Technol. 2005, 36, 976. 16. Richard, P.; Londesborough, J.; Putkonen, M.; Kalkkinen, N.; Penttila, M. J. Biol. Chem. 2001, 276, 40631. 17. Suzuki, T.; Tran, L. H.; Yogo, M.; Idota, O.; Kitamoto, N.; Kawai, K.; Takamizawa, K. J. Biosci. Bioeng. 2005, 100, 472. 18. Sullivan, R.; Zhao, H. Appl. Microbiol. Biotechnol. 2007, 77, 845. 19. Aarnikunnas, J. S.; Pihlajaniemi, A.; Palva, A.; Leisola, M.; Nyyssola, A. Appl. Environ. Microbiol. 2006, 72, 368. 20. Seiboth, B.; Hartl, L.; Pail, M.; Kubicek, C. P. Eukaryotic Cell 2003, 2, 867. 21. Tiwari, M.; Lee, J.-K. J. Mol. Graphics Modell. 2010, 28, 707. 22. Bae, B.; Sullivan, R. P.; Zhao, H.; Nair, S. K. J. Mol. Biol. 2011, 402, 230. 23. Gao, H.; Tiwari, M. K.; Kang, Y. C.; Lee, J.-K. Bioorg. Med. Chem. Lett. 2012, 22, 1931. 24. Tiwari, M. K.; Moon, H. J.; Jeya, M.; Lee, J.-K. Appl. Microbiol. Biotechnol. 2010, 87, 571. 25. Lee, J.-K.; Koo, B. S.; Kim, S. Y. Appl. Environ. Microbiol. 2003, 69, 6179. 26. Pail, M.; Peterbauer, T.; Seiboth, B.; Hametner, C.; Druzhinina, I.; Kubicek, C. P. Eur. J. Biochem. 2004, 271, 1864. 27. Kim, B.; Sullivan, R. P.; Zhao, H. Appl. Microbiol. Biotechnol. 2010, 87, 1407. 28. de Groot, M. J.; Prathumpai, W.; Visser, J.; Ruijter, G. J. Biotechnol. Prog. 2005, 21, 1610. 29. Kvassman, J.; Pettersson, G. Eur. J. Biochem. 1979, 100, 115. 30. Dickenson, C. J.; Dickinson, F. M. Biochem. J. 1975, 147, 303. 31. Klinman, J. P. J. Biol. Chem. 1975, 250, 2569. 32. Lindstad, R. I.; Koll, P.; McKinley-McKee, J. S. Biochem. J. 1998, 330, 479. 33. Lindstad, R. I.; McKinley-McKee, J. S. Eur. J. Biochem. 1995, 233, 891. 34. Tiwari, M. K.; Singh, R. K.; Singh, R.; Jeya, M.; Zhao, H.; Lee, J.-K. J. Biol. Chem. 2012, 287, 19429. 35. Karlsson, C.; Jornvall, H.; Hoog, J. O. Eur. J. Biochem. 1991, 198, 761. 36. Ng, K.; Ye, R.; Wu, X. C.; Wong, S. L. J. Biol. Chem. 1992, 267, 24989. 37. Sarthy, A. V.; Schopp, C.; Idler, K. B. Gene 1994, 140, 121. 38. Lindstad, R. I.; Hermansen, L. F.; McKinley-McKee, J. S. Eur. J. Biochem. 1994, 221, 847. 39. Pauly, T. A.; Ekstrom, J. L.; Beebe, D. A.; Chrunyk, B.; Cunningham, D.; Griffor, M.; Kamath, A.; Lee, S. E.; Madura, R.; McGuire, D.; Subashi, T.; Wasilko, D.; Watts, P.; Mylari, B. L.; Oates, P. J.; Adams, P. D.; Rath, V. L. Structure 2003, 11, 1071. 40. Doten, R. C.; Mortlock, R. P. Appl. Environ. Microbiol. 1985, 49, 158. 41. Khan, A. R.; Tokunaga, H.; Yoshida, K.; Izumori, K. J. Ferment. Bioeng. 1991, 72, 488.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
pH-rate profiles of L-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in L-xylulose production Manish Kumar Tiwari a,b,c, , Raushan Kumar Singh a, Hui Gao a, Taesu Kim a, Suhwan Chang d, Han S. Kim b,c,⇑, Jung-Kul Lee a,b,⇑ a
Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea Institute of SK-KU Biomaterials, Konkuk University, Seoul 143-701, Republic of Korea Department of Environmental Engineering, Konkuk University, Seoul 143-701, Republic of Korea d Department of Biomedical Sciences, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 June 2013 Revised 8 November 2013 Accepted 20 November 2013 Available online 27 November 2013 Keywords: Rare sugar L-Xylulose pH-Dependent kinetics Molecular dynamics
a b s t r a c t L-Arabinitol
4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of L-arabinitol oxidation by NAD+, catalyzed by HjLAD, was studied within the pH range of 7.0–9.5 at 25 °C. The turnover number (kcat) and the catalytic efficiency (kcat/ Km) were 4200 min 1 and 290 mM 1 min 1, respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare L-sugar L-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%. Ó 2013 Elsevier Ltd. All rights reserved.
L-Xylulose (threo-pentulose) is a ketopentose that is rare and expensive.1 In recent years, interest in L-carbohydrates and the L-nucleosides derived from them has substantially increased in medicine.2 Recent investigations have revealed that rare carbohydrates are potentially important for use as low-calorie carbohydrate sweeteners and bulking agents3,4 as well as potential inhibitors of various glycosidases.5,6 Moreover, the use of derivatives of an L-rare sugar as antihepatitis B virus agents has also been reported.7 Rare L-forms of sugars, such as L-xylose, have been used as starting materials for the synthesis of various pharmaceutically interesting molecules,8 including L-ribose, D-psicose, L-xylose, D-tagatose, and L-xylulose. Nucleoside analogues of L-sugars have potential as anticancer and cardioprotective agents, and they are being exploited as antiviral drugs against hepatitis-B and HIV.5,6 Recent reports suggest that novel L-xylose derivatives act as inhibitors of urinary glucose reabsorption, suggesting their application in diabetes treatment.9 Advancements in genomics, screening, and evolution technologies may lead to an increased availability of new and robust biocatalysts for industrial-scale application,
⇑ Corresponding authors. Tel.: +82 2 450 3505; fax: +82 2 458 3504 (H.S.K. and J.-K.L.). E-mail addresses: [email protected] (H.S. Kim), [email protected] (J.-K. Lee). Present address: Department of Chemistry, Technical University of Denmark, Kongens Lyngby 2800, Denmark. 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.047
and these advancements are stimulated by an increasing demand for catalysts that are used as ingredients.10 Biocatalysis often offers advantages over chemical synthesis, because enzyme-catalyzed reactions are often highly enantioselective and regioselective.10,11 These reactions can be conducted at an ambient temperature and atmospheric pressure, thereby avoiding the use of extreme conditions that can cause problems with isomerization, racemization, epimerization, and rearrangement.12 Therefore, enzymatic isomerization of the ketosugar L-xylulose to L-xylose is considered an alternative for chemical synthesis.13–15 The starting material L-xylulose can be produced by oxidation of a polyol substrate such as L-arabinitol by using recombinant catalysts.16–18 An efficient production method for L-xylulose would allow utilization of this rare sugar in new applications of commercial value.19L-Arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12), a common enzyme found in yeasts and filamentous fungi, catalyzes the second step of the fungal L-arabinose metabolic pathway by oxidizing L-arabinitol to
Figure 1. Bioconversion of L-arabinitol into L-xylulose using LAD.
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(Fig. 1) with concomitant NAD+ reduction.16,20 Both are intermediates in catabolic pathways for pentose and pentitol utilization in many organisms.19 This enzyme is known to operate by an effectively ordered ternary complex mechanism, in which coenzyme-binding precedes substrate binding.21 The stoichiometry of the overall reaction prescribes that a proton is liberated for every NADH molecule formed, indicating that the catalytic reaction is critically dependent on one or several proton transfer steps. Such proton transfers are expected to involve ionizing groups at the active center of the enzyme.22 Few studies have reported the kinetic parameters of LADs, and the reported values were largely dependent on experimental conditions such as the pH and buffer used. However, pH dependence of LAD has not been thoroughly investigated to date, and important aspects of pH-dependence and its relationship with kinetic behavior of LAD remain unclear. In addition, to obtain rare sugars on an industrial scale, a less-expensive production method with high yield is required. Therefore, it is highly desirable to identify conditions for LAD enzyme that can exhibit high catalytic efficiency. In the present Letter, we have reported pH-dependent kinetics of fungal LAD from Hypocrea jecorina (HjLAD) and its application in the production of L-xylulose. In an attempt to investigate the pH-dependent kinetics of HjLAD and the production of L-xylulose, the HjLAD gene was cloned into the T7 promoter-based plasmid pET28a to form pET28a-HjLAD, and it was then heterologously expressed23 in Escherichia coli BL21 (DE3) (see Supplementary data). The enzyme was purified using Ni-nitrilotriacetic acid affinity chromatography (Fig. 2), and the purified HjLAD showed high activity toward L-arabinitol oxidation. Using gel filtration chromatography on the Sephacryl S-300 High Resolution media (column 16/60; Amersham, UK), we found that HjLAD corresponded to a molecular weight of approximately 174 kDa (Fig. S1). The subunit molecular weight of the enzyme was approximately 41 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 2). These results suggest that HjLAD is a noncovalently linked tetramer in its native form. HjLAD showed activity in the pH range of 7.0–10.5, with the L-xylulose
L-and L-xyluloses
Figure 2. Overexpression and purification of recombinant HjLAD. Lane M, the molecular weight marker proteins (kDa). Lane 1, whole-cell fraction of recombinant HjLAD. Lane 2, insoluble protein fraction. Lane 3, 10 lg of soluble protein fraction of recombinant BL21 (DE3/pET28a-HjLAD). Lane 4, 4 lg of purified HjLAD enzyme.
maximum activity observed at pH 9.5 (Fig. S2). The enzyme activity was significantly reduced above pH 9.5 and below pH 7.0, and approximately 10% and 40% of the enzyme activity was retained at pH 7.0 and 10.5, respectively (Fig. S2). However, NAD+ appeared to be unstable at pH values above 10.5 during extended incubation.19,24 Therefore, enzymatic activities in this study were routinely determined in the pH range of 7.0–9.5. The optimal temperature for oxidation of L-arabinitol was 65 °C (Fig. S3). Under optimum assay conditions, the kinetics of L-arabinitol oxidation, catalyzed by LAD, were studied in the pH range of 7.0–9.5 at 25 °C. The reactions were conducted in 100 mM Tris–HCl (pH 7.0–8.0) and Tris–glycine–NaOH (pH 8.5–9.5). Initial rates were determined by measuring changes in absorbance at 340 nm by using an ultraviolet (UV)–visible spectrophotomer at room temperature.24 Various concentrations of the substrate (L-arabinitol; 25– 500 mM) and cofactors (NAD+; 0.25–2 mM) were used to determine the kinetic parameters. Enzyme kinetics for the substrate and cofactors were analyzed using Michaelis–Menten kinetics, and the kinetic parameters were determined by fitting data to the non-linear regression plot.24 The catalytic activity of HjLAD is strongly dependent on pH over the pH range of 7.0–9.5 (Table 1). Maximal HjLAD activity at an alkaline pH for alcohol dehydrogenation of sugar is a common feature of similar enzymes isolated from diverse microbial systems.24,25 Specific activity of HjLAD was reported to be between 0.013 and 1.6 U/mg;16,26 these values were obtained with partially purified protein or a subsaturated cofactor concentration (0.25 mM) with purified enzyme. The specific activity of Trichoderma longibrachiatum LAD reported by Kim et al. using recombinant His-tagged purified protein was 8.7 U/mg at pH 7.0.27 In this study, at pH 7.0, Km of HjLAD was 18.2 ± 1.2 mM for L-arabinitol and 0.20 ± 0.03 mM for NAD+; these values were consistent with those reported in the literature.16,26,27 However, at pH 9.5, the specific activity of HjLAD was 105 ± 9 U/mg-protein, which is significantly higher than that previously reported, and Km of HjLAD was 14.5 ± 1.0 mM for L-arabinitol, which is slightly lower than that reported in previous studies, thereby resulting in much higher catalytic efficiency (kcat/Km = 290; Table 1). It is clear from the results in Table 1 and Fig. 3 that the catalytic rate of HjLAD was essentially dependent on the pH range 7.0–9.5. At pH 7.0, the kcat value of HjLAD enzyme decreased to approximately 22% of that at pH 9.5. Table 2 shows a comparison of the properties of various LADs from different sources. It is noteworthy that HjLAD shows the highest turnover rate (kcat) and catalytic efficiency (kcat/Km) among all previously characterized LADs. The kcat and kcat/Km of HjLAD were 1760 min 1 and 102 mM 1 min 1, and 4200 min 1 and 290 mM 1 min 1 at pH 8.0 and 9.5, respectively. It has been established that this pH dependence is derived from the combined effects of pH on several reaction steps in a catalytic mechanism; proton equilibria involving ionizing groups on the enzyme are known to affect the binding of both coenzyme and substrate, as well as the inter-conversion of the productive ternary complexes.29 Based on the study on pH-dependence of the reactions of yeast alcohol dehydrogenase, Dickenson et al. proposed
Table 1 Kinetic properties of HjLAD enzymes on various pH range 7.0 to 9.5 pH
Specific activity (U/mg)
9.5 9.0 8.6 8.0 7.5 7.0
105 ± 9 99 ± 5 96 ± 7 44 ± 2 36 ± 2 24 ± 1
Km (mM)
kcat/Km, (mM
Coenzyme
Substrate
0.50 ± 0.08 0.40 ± 0.08 0.40 ± 0.05 0.50 ± 0.02 0.20 ± 0.07 0.20 ± 0.08
14.5 ± 1.0 14.2 ± 1.0 17.8 ± 1.3 17.2 ± 1.6 16.4 ± 2.3 18.2 ± 1.2
289 ± 32 278 ± 30 215 ± 25 102 ± 13 87.8 ± 17.4 52.7 ± 10.1
1
min
1
)
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Figure 3. pH dependence of the turnover of enzymatic L-arabinitol oxidation.
that the transition state for alcohol oxidation is favored by the presence of a basic group in the vicinity of the hydroxyl group of the alcohol substrate. This basic group becomes protonated when hydride transfer occurs.30 However, the uptake of the proton in the course of the forward reaction can occur directly from the solvent or, alternatively, a catalytic residue at the enzyme active site can function in acid–base catalysis.31 Studies on steady-state kinetics of sheep live sorbitol dehydrogenase suggest that the kinetic constants vary with pH in accordance with a single pK of 7.1 for sorbitol oxidation.32 Furthermore, the pK value was assigned to the ionization properties of a catalytically essential group, and the pK values found for sorbitol oxidation referred to a histidine residue.33 In the present study, to depict the microenvironment of HjLAD active site, we derived a computational structure of HjLAD and docked with L-arabinitol.21,34 This structure was modeled based on a recently reported crystal structure of LAD from Neurospora crassa22 (NcLAD; Protein Data Bank accession No.: 3M6I), which shares 80% of the sequence identity with that of HjLAD.34 HjLAD showed a similar residual positioning; the Cys66, His91, Glu92, and Glu176 residues were involved in coordination of catalytic Zn2+ along with a water molecule (Fig. 4). Sequence alignment between HjLAD and other homologous protein sequences coupled with molecular dynamics suggests that the His91 is conserved in the more distant members of the alcohol/sorbitol dehydrogenase family. His91 in HjLAD (Fig. 4) is present in SDHs, including the liver enzyme from rat, sheep, and humans,35 as well as in Bacillus subtilis36 and yeast SDH,37
Figure 4. Homology modeling and docking of L-arabinitol in the substrate-binding pocket of HjLAD suggesting routes of hydride transfer, where the key amino acid residues comprise the core region. Carbon atoms of active-site residues and nicotinamide ring are in white; carbon atoms in L-arabinitol are in green. Nitrogen, oxygen, and sulfur atoms are in blue, red, and yellow, respectively.
and it is equivalent to His68 in sheep liver alcohol dehydrogenase38 and His70 in human sorbitol dehydrogenase.39 On the basis of the identity of the catalytic microenvironment at the active site of HjLAD and related enzymes, one may hope to observe similarities in the reaction at the catalytic center. Furthermore, the indispensable role of His91 in catalysis was confirmed by site-directed mutagenesis. H91A mutant enzyme was successfully overexpressed with bands displaying a molecular mass of approximately 40 kDa, as expected for a monomer of LAD (Fig. S4A). The secondary structure of the H91A was analyzed along with the wild-type HjLAD.34 The wild-type enzyme and the purified protein from H91A mutant exhibited similar circular dichroism (CD) spectra, with ellipticity minima of comparable amplitude in the range of 220–240 nm. The CD spectra of all of the native proteins were very similar (Fig. S4B). This indicated that all tested wild-type and mutant enzymes had a similar secondary structure and that the global folding of mutant and wild-type HjLAD is similar. However, despite its efficient expression and similar secondary structure, the HjLAD mutant (H91A) was inactive, which demonstrates its crucial role in catalysis. Using thermostable27 and highly active biocatalyst HjLAD, L-xylulose production was carried out at pH 9.5 and 7.0 (see Supplementary data). In the present study, L-arabinitol, a naturally occurring polyol that is commercially available, was used as the
Table 2 Biochemical and kinetic properties of LAD enzymes from various organisms Organism
Hypocrea jecorina Aspergillu oryzae Neurospora crassa Hypocrea jecorina Aspergillus niger Trichoderma longibrachiatum Penicillium chrysogenum Aspergillus niger Hypocrea jecorina Hypocrea jecorina Hypocrea jecorina NR: not reported.
Optimum pH
Kinetics pH
NR NR 9.5 NR 9.4 9.4
7.0 NR 8.0 7.0 7.0 7.0
9.4 NR 9.5 9.5 9.5
7.0 9.6 7.0 8.0 9.5
Specific activity (U/mg) 1.6 0.04 31 0.01 11.7 8.7 25.3 96 24 44 105
Km (mM)
kcat (min
1
)
kcat/Km, Larabinitol (mM 1 min
References
Coenzyme (NAD+)
Substrate (L-arabinitol)
0.18 NR 0.17 NR 0.2 0.2
40 NR 16 4.5 25 18
NR NR 1206 51 507 346
NR NR 75 11 20 19
16 17 18 26 27 27
0.3 0.5 0.2 0.5 0.2
37 89 18.2 17.2 14.5
1085 NR 960 1760 4200
29 NR 53 102 290
27 28 This study This study This study
1
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(Grant 2011-50210). This work was also supported by the NRF Grant funded by the Korea government (2012M3C5A1053339). This work was supported by a Grant from the Next-Generation BioGreen 21 program (No. PJ009605), Rural Development Administration, Republic of Korea. This research was also supported by the 2012 KU Brain Pool of Konkuk University. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 11.047. References and notes
Figure 5. Time course production of L-xylulose using recombinant HjLAD at pH 9.5 (j) and pH 7.0 (N). The error bars show the standard deviation for three determinations.
starting material. The time courses of L-xylulose production at pH 9.5 and 7.0 are presented in Fig. 5. In the reaction mixture initially containing 5 g l 1 L-arabinitol, the conversion of L-arabinitol to Lxylulose reached 86 ± 3% and a volumetric productivity of 2.2 g l 1 h 1 was obtained after 2 h of reaction at pH 9.5. While at pH 7.0, L-xylulose productivity was significantly decreased. After 2 h of reaction, only 42 ± 4% conversion was obtained. In the control experiment without LAD enzyme, no L-arabinitol consumption or L-xylulose production was detected during 4 h of incubation of the reaction mixture. The current production study is favorably comparable with L-xylulose production rates reported in the literature.19,40 The highest L-xylulose yields from xylitol of 70% for Alcaligenes sp. strain 701B41 and 80% for Pantoea ananatis ATCC 4307219 were obtained at a xylitol concentration of 5 g l 1 in 24 and 18 h, respectively. A yield of approximately 86% was achieved within 2 h with the recombinant purified HjLAD in the present study. We further purified L-xylulose using ion-exchange column chromatography (see Supplementary data). As a result, we were able to obtain 99% pure L-xylulose (3.9 g) from L-arabinitol (5 g) in 78% yield. The purified L-xylulose was characterized by high resolution electrospray ionization mass spectrometry (HR-ESI MS) and 1 H NMR spectroscopy (see Supplementary data text, Figs. S5 and S6). The HR-ESI-MS (negative-ion mode [M–H] ) revealed a molecular weight of 149.0280 corresponding to the molecular formula of L-xylulose, C5H10O5. In summary, the gene (HjLAD) encoding LAD was cloned from H. jecorina and overexpressed in a soluble form. The purified HjLAD protein exhibited the highest LAD activity. By analogy with other reductases/dehydrogenases that share similarity at the sequence level, we speculated that the conserved His91, a potential activesite acid residue, could function in acid–base catalysis. To the best of our knowledge, this is the first study demonstrating the feasibility of LAD (HjLAD) in the production of rare sugars such as L-xylulose, which gave a yield of approximately 86% in 2 h. Therefore, HjLAD can be effectively utilized in biotechnological production of this value-added rare sugar. Acknowledgments This research was supported by the Converging Research Center Program through the National Research Foundation of Korea
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