- Email: [email protected]
Identification of malic enzyme mutants depending on 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide analogs
Bioorganic & Medicinal Chemistry Letters 24 (2014) 1307–1309
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification of malic enzyme mutants depending on 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide analogs Shuhua Hou a,b, Debin Ji a, Wujun Liu a, Lei Wang a, Zongbao (Kent) Zhao a,⇑ a b
Division of Biotechnology and State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China Department of Chemistry, Liaoning Key Laboratory for the Synthesis and Application of Functional Compounds, Bohai University, Jinzhou 121013, China
a r t i c l e
i n f o
Article history: Received 17 December 2013 Revised 15 January 2014 Accepted 20 January 2014 Available online 28 January 2014 Keywords: Cofactor Malic enzyme Nicotinamide adenine dinucleotide Orthogonal redox system Enzyme kinetics
a b s t r a c t An activity screening between 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide (NAD) analogs and malic enzyme (ME) mutants identified some mutants capable of taking NAD analogs as the cofactor. One particular pair, ME-L310K/L404S and the analog B-8 had good catalytic efficiency and cofactor specificity. The new system gained about 1200-fold cofactor specificity shift from NAD toward B-8 in terms of oxidative decarboxylation of L-malate. Our results provided insightful information for the development of orthogonal redox system that is of particular important to precisely control engineered metabolic pathways. Ó 2014 Elsevier Ltd. All rights reserved.
Nicotinamide adenine dinucleotide (NAD, Fig. 1) is one of the most important cofactors in life.1,2 It behaves not only as cofactor for redox enzymes but also mediates other events such as calcium homeostasis, gene expression, carcinogenesis, aging and cell death.3,4 Any perturbations leading to NAD concentration fluctuation are deemed to have major global effects because cellular processes are tightly connected to NAD.5,6 Thus, it remains challenging to manipulate NAD-dependent reactions at the cofactor level. Bioorthogonal chemistry has received major attentions partially because abiotic molecules can work in living systems with less interference with the surrounding biological milieu.7–9 Many bioorthogonal reactions have been developed and successfully applied in addressing difficult biological problems.10–13 In order to isolate the targeted NAD-dependent reaction from other NAD-dependent biological processes, we recently proposed to devise bioorthogonal redox systems depending on synthetic cofactors. A prototype of such system was demonstrated by creating active malic enzyme (ME) mutants depending on nicotinamide flucytosine dinucleotide (NFCD) and nicotinamide cytosine dinucleotide (NCD).14 The mutant ME-L310R/Q401C showed excellent activity with NFCD among other NAD analogs, whereas marginal activity with NAD.14,15 Due to its independence of endogenous cofactor regeneration, the engineered redox systems opened the window for metabolic engineering to control production routes with precise ‘first-time-right’ predictions.16 To further explore the chemical space of redox ⇑ Corresponding author. Tel./fax: +86 411 8437 9211. E-mail address: [email protected] (Z. K. Zhao). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2014.01.047
cofactors, we have synthesized 1,2,3-triazole moiety-containing NAD analogs (Fig. 1), and observed only marginal activities for recombinant wild-type ME in the presence of these analogs.17 It should be noted that the low levels of activity are favored in evolution, such that the mutant enzyme might be turned into a much more proficient catalyst.18,19 However, it remains to be demonstrated whether ME mutants can be found to take these analogs as effective cofactors. In this work, we assayed these 1,2,3-triazole moiety-containing NAD analogs against ME mutants that showed low activities using NAD as the cofactor as shown in our previous study.14 Two pairs of NAD analog/ME mutant were identified that had relatively high catalytic efficiencies. These results provided additional examples of synthetic redox catalysis and further suggested that molecular interactions between cofactor and redox enzymes can be engineered to enable abiotic compound-mediated biocatalysis. We first selected three representative analogs, B-3, B-8 and B-13, to screen for activity against twelve mutant MEs purified as early reported20 (Table 1). These three analogs covered good structural diversities of the analog library in terms of the size and polarity of the substituent attached to the 1,2,3-triazole ring. Such structural diversities were important to generate new interactions to match the cofactor binding sites of the mutant enzymes. The specific activity was determined with a UV–vis spectrophotometer (Supporting Information). No catalytic activity was detected for four ME mutants, L310R/Q401C, L310R/Q401D L310R/ Q401G and L310R/L404I, in the presence of these analogs. For B-13, ten mutants were inactive and only ME-L310K/Q401R and
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S. Hou et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1307–1309
O
OH
HO
O N
N
N
O
NH2
O
OH O
R
O
+
OH
HO
NAD
HO
N
N
O
O-
OH
N
N
NH2
O
O P O P O
NH2
O
O P O P O
N
O-
+
N
O
OHO
OH
F3 C
F OH
R= B-1
OMe B-2
F
B-3
B-4
B-5
N B-8
O O-
NH2 B-11
B-10
B-9
O
NH
CH3
B-7
B-6
O
O NH
H3 C
B-12
B-13
Figure 1. Structures of NAD and 1,2,3-triazole moiety-containing analogs.
ME-L310K/L404S showed activity. The size of the substituted 1,2,3triazole moiety of B-13 should be much smaller than that of the adenosine moiety of NAD. Because the cofactor binding pockets of most of these double mutants likely had similar size to that of ME, it was reasonable that B-13 failed to fit well. However, the presence of the carboxylic acid group in B-13 might help charge interaction as indicated for ME-L310K/Q401R. For B-3, seven of the twelve mutant enzymes had low activity, while for B-8, six showed activity. It should be noted that five ME mutants had activity in the presence of B-3 and B-8, indicating that similar interactions might be involved by the p-methoxyphenyl group and the 2-naphthalenyl group, respectively. Among these functional pairs, the mutant ME-L310K/L404S and B-8 was the most active one and had a specific activity of 10.5 U mg1. The fact that MEL310K/L404S showed reasonably good specific activity in the presence of B-8 suggested that this mutant might be more suitable for 1,2,3-triazole moiety-containing NAD analogs. Thus, we tested other NAD analogs listed in Figure 1 to see whether they could be recognized by ME-L310K/L404S. Fortunately, similar activity was found using B-7 as coenzyme, and lower activity was observed
for the analog B-2. Compared with B-7 and B-8, B-2 had an intact phenyl group attached to the 1,2,3-triazole ring. To understand more details of the catalysis, we determined kinetic parameters of ME and ME-L310K/L404S using NAD, B-2, B-7 and B-8, respectively, as cofactor. Figure 2 shows the time course of ME-L310K/L404S catalyzed reaction monitored by evolution of reduced form of B-8 at 340 nm. Initial reaction rates of MEL310K/L404S were measured in the presence of different concentrations of B-8. The Lineweaver–Burk plot yielded a straight line as shown in the insert of Figure 2. It demonstrated that the kinetics data fit well with Michaelis–Menten equation. Similarly, a comprehensive kinetic dataset toward NAD and analogs were obtained (Table 2). For wild type ME, the kcat value in the presence of NAD was more than 10-fold higher than those in the presence of NAD analogs, while the KM value for NAD was less than 20% of those for NAD analogs. Thus, the kcat/KM value for the ME and NAD
0.9
0.8
Table 1 Screen results and the specific activity of ME mutants with B-3, B-8 and B-13
a
0.7
B-3
B-8
B-13
ND ND ND 0.98 0.43 4.3 0.43 ND 0.51 ND 0.51 1.6
ND ND ND ND 0.32 2.3 ND 0.53 0.36 ND 0.47 10.5
ND ND ND ND ND 2.1 ND ND ND ND ND 0.17
The specific activity was determined, while the concentration of NAD analogs was at 0.2 mM. ND was not detected.
1/Specific activity (mg/U
ME-L310R/Q401C ME-L310R/Q401D ME-L310R/Q401G ME-L310R/Q401I ME-L310R/Q401N ME-L310K/Q401R ME-L310R/Q401S ME-L310R/Q401V ME-L310R/L404C ME-L310R/L404I ME-L310R/L404N ME-L310K/L404S
Specific activitya (U mg1)
A340
ME mutants
0.6
0.5 1/[B-8] (1/mM)
0.4 -20
0
20
40
60
80
100
120
140
160
Time (s) Figure 2. Time course of the formation of the reduced form of B-8 by ME-L310K/ L404S in the presence of L-malate. Insert: Lineweaver–Burk plot.
1309
S. Hou et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1307–1309 Table 2 Kinetic parameters for ME and ME-L310K/L404S using NAD, B-2, B-7 or B-8 as the cofactor NAD analogs
ME kcat (s
NAD B-2 B-7 B-8
ME-L310K/L404S 1
)
103.2 ± 3.7 7.5 ± 1.4 10.0 ± 0.23 4.6 ± 0.69
1
KM (mM)
kcat/KM (mM
0.28 ± 0.021 2.9 ± 0.70 1.6 ± 0.055 1.8 ± 0.34
368.6 2.6 6.3 2.6
1
s
)
system was about two orders of magnitude higher than those of the ME and NAD analog systems, indicating that these analogs were strongly disfavored by ME. Compared to wild type ME, the kcat value for ME-L310K/L404S in the presence of NAD dropped by approximately 70% to 33.7 s1, and the KM value increased by about 15-fold to 4.1 mM. Thus, ME-L310K/L404S bound NAD substantially weaker and retained only 2.2% catalytic efficiency of that of ME, indicating that NAD was strongly disfavored by the mutant enzyme. It should be emphasized that such a low catalytic efficiency was in fact beneficial to develop orthogonal systems depending on cofactor analogs. For ME-L310K/L404S, both kcat and KM were lower in the presence of B-2 that those in the presence of NAD. As a result, MEL310K/L404S had a very close catalytic efficiency with B-2 to that with NAD, suggesting that there was little cofactor preference between these two compounds and that B-2 did not match well with the mutant enzyme. Fortunately, ME-L310K/L404S had lower KM values of 1.1 mM and 1.0 mM, respectively, for analog B-7 and B-8, indicating that these two compounds fit much better in the engineered cofactor binding pocket. These data also suggested that ME-L310K/L404S might have a spacious cofactor binding site for NAD analogs with bulkier groups attached to the 1,2,3-triazole ring. Moreover, ME-L310K/L404S showed much higher kcat values in the presence of B-7 or B-8. As a result, catalytic efficiency of ME-L310K/L404S improved by more 10-fold to 78.8 mM1 s1 and 68.6 mM1 s1, respectively, upon switching the cofactor from B-2 to B-7 or B-8. The ratio between the kcat/KM value of ME-L310K/L404S and that of ME for each cofactor indicated the tendency of the corresponding cofactor favored by the mutant enzyme. Thus, the ratio was 0.022 in the case of NAD, indicating NAD was strongly disfavored by ME-L310K/L404S. Whereas, for these three NAD analogs, the ratios were higher than 1, indicating these analogs were favored by ME-L310K/L404S. Importantly, for enzymatic catalyzed oxidative decarboxylation of L-malate, the engineered system gained approximately 120-, 570- and 1200-fold cofactor specificity shift from NAD toward B-2, B-7 and B-8, respectively. Thus, the system ME-L310K/L404S and B-8 had the most stringent orthogonality to the natural system using ME and NAD. It should be noted that cofactor specificity for natural NAD(P)-dependent enzymes varied substantially in favoring of NAD or NADP.21–23 ME-L310K/ L404S actually had better cofactor specificity for B-8 than many natural enzymes for NAD or NADP. In summary, an activity screening between NAD analogs and ME mutants led to the identification of some mutants capable of
kcat (s
1
)
33.7 ± 1.9 16.4 ± 1.1 86.7 ± 6.6 68.6 ± 5.6
ðkcat =K M ÞMEL310K=L404S ðkcat =K M ÞME
KM (mM)
kcat/KM (mM
4.1 ± 0.42 2.5 ± 0.22 1.1 ± 0.12 1.0 ± 0.11
8.2 6.7 78.8 68.6
1
s
1
) 0.022 2.58 12.5 26.4
taking NAD analogs as the cofactor. One particular pair, MEL310K/L404S and the analog B-8 had good catalytic efficiency and cofactor specificity. Our results provided insightful information for the development of orthogonal redox system that is of particular important to precisely control engineered metabolic pathway in the biological systems.16 Acknowledgments This work was supported by the National Basic Research and Development Program of China (No. 2012CB721103) and National Natural Science Foundation of China (Nos. 21102143, 21325627). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl. 2014. 01.047. References and notes 1. Blank, L. M.; Ebert, B. E.; Buehler, K.; Bühler, B. Antioxid. Redox Signal 2010, 13, 349. 2. Pollak, N.; Dölle, C.; Ziegler, M. Biochem. J. 2007, 402, 205. 3. Ying, W. Antioxid. Redox Signal 2008, 10, 179. 4. Houtkooper, R. H.; Cantó, C.; Wanders, R. J.; Auwerx, J. Endocr. Rev. 2010, 31, 194. 5. Yang, H.; Yang, T.; Baur, J. A.; Perez, E.; Matsui, T.; Carmona, J. J.; Lamming, D. W.; Souza-Pinto, N. C.; Bohr, V. A.; Rosenzweig, A. Cell 2007, 130, 1095. 6. Holm, A. K.; Blank, L. M.; Oldiges, M.; Schmid, A.; Solem, C.; Jensen, P. R.; Vemuri, G. N. J. Biol. Chem. 2010, 285, 17498. 7. Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 651. 8. Boyce, M.; Bertozzi, C. R. Nat. Methods 2011, 8, 638. 9. Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13. 10. Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666. 11. McLachlan, M. J.; Chockalingam, K.; Lai, K. C.; Zhao, H. Angew. Chem., Int. Ed. 2009, 48, 7783. 12. Raghavan, A. S.; Hang, H. C. Drug Discovery Today 2009, 14, 178. 13. Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974. 14. Ji, D.; Wang, L.; Hou, S.; Liu, W.; Wang, J.; Wang, Q.; Zhao, Z. K. J. Am. Chem. Soc. 2011, 133, 20857. 15. Ji, D.; Wang, L.; Liu, W.; Hou, S.; Zhao, K. Z. Sci. China Chem. 2013, 56, 296. 16. Mampel, J.; Buescher, J.; Meurer, G.; Eck, J. Trends Biotechnol. 2013, 31, 52. 17. Hou, S.; Liu, W.; Ji, D.; Wang, Q.; Zhao, Z. K. Tetrahedron Lett. 2011, 52, 5855. 18. Tracewell, C. A.; Arnold, F. H. Curr. Opin. Chem. Biol. 2009, 13, 3. 19. Nobeli, I.; Favia, A. D.; Thornton, J. M. Nat. Biotechnol. 2009, 27, 157. 20. Wang, J.; Zhang, S.; Tan, H.; Zhao, Z. K. J. Microbiol. Methods 2007, 71, 225. 21. Steen, I. H.; Lien, T.; Madsen, M. S.; Birkeland, N.-K. Arch. Microbiol. 2002, 178, 297. 22. Capone, M.; Scanlon, D.; Griffin, J.; Engel, P. C. FEBS J. 2011, 278, 2460. 23. Hsieh, Y.-J.; Hung, H.-C. J. Biol. Chem. 2009, 284, 4536.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification of malic enzyme mutants depending on 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide analogs Shuhua Hou a,b, Debin Ji a, Wujun Liu a, Lei Wang a, Zongbao (Kent) Zhao a,⇑ a b
Division of Biotechnology and State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China Department of Chemistry, Liaoning Key Laboratory for the Synthesis and Application of Functional Compounds, Bohai University, Jinzhou 121013, China
a r t i c l e
i n f o
Article history: Received 17 December 2013 Revised 15 January 2014 Accepted 20 January 2014 Available online 28 January 2014 Keywords: Cofactor Malic enzyme Nicotinamide adenine dinucleotide Orthogonal redox system Enzyme kinetics
a b s t r a c t An activity screening between 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide (NAD) analogs and malic enzyme (ME) mutants identified some mutants capable of taking NAD analogs as the cofactor. One particular pair, ME-L310K/L404S and the analog B-8 had good catalytic efficiency and cofactor specificity. The new system gained about 1200-fold cofactor specificity shift from NAD toward B-8 in terms of oxidative decarboxylation of L-malate. Our results provided insightful information for the development of orthogonal redox system that is of particular important to precisely control engineered metabolic pathways. Ó 2014 Elsevier Ltd. All rights reserved.
Nicotinamide adenine dinucleotide (NAD, Fig. 1) is one of the most important cofactors in life.1,2 It behaves not only as cofactor for redox enzymes but also mediates other events such as calcium homeostasis, gene expression, carcinogenesis, aging and cell death.3,4 Any perturbations leading to NAD concentration fluctuation are deemed to have major global effects because cellular processes are tightly connected to NAD.5,6 Thus, it remains challenging to manipulate NAD-dependent reactions at the cofactor level. Bioorthogonal chemistry has received major attentions partially because abiotic molecules can work in living systems with less interference with the surrounding biological milieu.7–9 Many bioorthogonal reactions have been developed and successfully applied in addressing difficult biological problems.10–13 In order to isolate the targeted NAD-dependent reaction from other NAD-dependent biological processes, we recently proposed to devise bioorthogonal redox systems depending on synthetic cofactors. A prototype of such system was demonstrated by creating active malic enzyme (ME) mutants depending on nicotinamide flucytosine dinucleotide (NFCD) and nicotinamide cytosine dinucleotide (NCD).14 The mutant ME-L310R/Q401C showed excellent activity with NFCD among other NAD analogs, whereas marginal activity with NAD.14,15 Due to its independence of endogenous cofactor regeneration, the engineered redox systems opened the window for metabolic engineering to control production routes with precise ‘first-time-right’ predictions.16 To further explore the chemical space of redox ⇑ Corresponding author. Tel./fax: +86 411 8437 9211. E-mail address: [email protected] (Z. K. Zhao). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2014.01.047
cofactors, we have synthesized 1,2,3-triazole moiety-containing NAD analogs (Fig. 1), and observed only marginal activities for recombinant wild-type ME in the presence of these analogs.17 It should be noted that the low levels of activity are favored in evolution, such that the mutant enzyme might be turned into a much more proficient catalyst.18,19 However, it remains to be demonstrated whether ME mutants can be found to take these analogs as effective cofactors. In this work, we assayed these 1,2,3-triazole moiety-containing NAD analogs against ME mutants that showed low activities using NAD as the cofactor as shown in our previous study.14 Two pairs of NAD analog/ME mutant were identified that had relatively high catalytic efficiencies. These results provided additional examples of synthetic redox catalysis and further suggested that molecular interactions between cofactor and redox enzymes can be engineered to enable abiotic compound-mediated biocatalysis. We first selected three representative analogs, B-3, B-8 and B-13, to screen for activity against twelve mutant MEs purified as early reported20 (Table 1). These three analogs covered good structural diversities of the analog library in terms of the size and polarity of the substituent attached to the 1,2,3-triazole ring. Such structural diversities were important to generate new interactions to match the cofactor binding sites of the mutant enzymes. The specific activity was determined with a UV–vis spectrophotometer (Supporting Information). No catalytic activity was detected for four ME mutants, L310R/Q401C, L310R/Q401D L310R/ Q401G and L310R/L404I, in the presence of these analogs. For B-13, ten mutants were inactive and only ME-L310K/Q401R and
1308
S. Hou et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1307–1309
O
OH
HO
O N
N
N
O
NH2
O
OH O
R
O
+
OH
HO
NAD
HO
N
N
O
O-
OH
N
N
NH2
O
O P O P O
NH2
O
O P O P O
N
O-
+
N
O
OHO
OH
F3 C
F OH
R= B-1
OMe B-2
F
B-3
B-4
B-5
N B-8
O O-
NH2 B-11
B-10
B-9
O
NH
CH3
B-7
B-6
O
O NH
H3 C
B-12
B-13
Figure 1. Structures of NAD and 1,2,3-triazole moiety-containing analogs.
ME-L310K/L404S showed activity. The size of the substituted 1,2,3triazole moiety of B-13 should be much smaller than that of the adenosine moiety of NAD. Because the cofactor binding pockets of most of these double mutants likely had similar size to that of ME, it was reasonable that B-13 failed to fit well. However, the presence of the carboxylic acid group in B-13 might help charge interaction as indicated for ME-L310K/Q401R. For B-3, seven of the twelve mutant enzymes had low activity, while for B-8, six showed activity. It should be noted that five ME mutants had activity in the presence of B-3 and B-8, indicating that similar interactions might be involved by the p-methoxyphenyl group and the 2-naphthalenyl group, respectively. Among these functional pairs, the mutant ME-L310K/L404S and B-8 was the most active one and had a specific activity of 10.5 U mg1. The fact that MEL310K/L404S showed reasonably good specific activity in the presence of B-8 suggested that this mutant might be more suitable for 1,2,3-triazole moiety-containing NAD analogs. Thus, we tested other NAD analogs listed in Figure 1 to see whether they could be recognized by ME-L310K/L404S. Fortunately, similar activity was found using B-7 as coenzyme, and lower activity was observed
for the analog B-2. Compared with B-7 and B-8, B-2 had an intact phenyl group attached to the 1,2,3-triazole ring. To understand more details of the catalysis, we determined kinetic parameters of ME and ME-L310K/L404S using NAD, B-2, B-7 and B-8, respectively, as cofactor. Figure 2 shows the time course of ME-L310K/L404S catalyzed reaction monitored by evolution of reduced form of B-8 at 340 nm. Initial reaction rates of MEL310K/L404S were measured in the presence of different concentrations of B-8. The Lineweaver–Burk plot yielded a straight line as shown in the insert of Figure 2. It demonstrated that the kinetics data fit well with Michaelis–Menten equation. Similarly, a comprehensive kinetic dataset toward NAD and analogs were obtained (Table 2). For wild type ME, the kcat value in the presence of NAD was more than 10-fold higher than those in the presence of NAD analogs, while the KM value for NAD was less than 20% of those for NAD analogs. Thus, the kcat/KM value for the ME and NAD
0.9
0.8
Table 1 Screen results and the specific activity of ME mutants with B-3, B-8 and B-13
a
0.7
B-3
B-8
B-13
ND ND ND 0.98 0.43 4.3 0.43 ND 0.51 ND 0.51 1.6
ND ND ND ND 0.32 2.3 ND 0.53 0.36 ND 0.47 10.5
ND ND ND ND ND 2.1 ND ND ND ND ND 0.17
The specific activity was determined, while the concentration of NAD analogs was at 0.2 mM. ND was not detected.
1/Specific activity (mg/U
ME-L310R/Q401C ME-L310R/Q401D ME-L310R/Q401G ME-L310R/Q401I ME-L310R/Q401N ME-L310K/Q401R ME-L310R/Q401S ME-L310R/Q401V ME-L310R/L404C ME-L310R/L404I ME-L310R/L404N ME-L310K/L404S
Specific activitya (U mg1)
A340
ME mutants
0.6
0.5 1/[B-8] (1/mM)
0.4 -20
0
20
40
60
80
100
120
140
160
Time (s) Figure 2. Time course of the formation of the reduced form of B-8 by ME-L310K/ L404S in the presence of L-malate. Insert: Lineweaver–Burk plot.
1309
S. Hou et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1307–1309 Table 2 Kinetic parameters for ME and ME-L310K/L404S using NAD, B-2, B-7 or B-8 as the cofactor NAD analogs
ME kcat (s
NAD B-2 B-7 B-8
ME-L310K/L404S 1
)
103.2 ± 3.7 7.5 ± 1.4 10.0 ± 0.23 4.6 ± 0.69
1
KM (mM)
kcat/KM (mM
0.28 ± 0.021 2.9 ± 0.70 1.6 ± 0.055 1.8 ± 0.34
368.6 2.6 6.3 2.6
1
s
)
system was about two orders of magnitude higher than those of the ME and NAD analog systems, indicating that these analogs were strongly disfavored by ME. Compared to wild type ME, the kcat value for ME-L310K/L404S in the presence of NAD dropped by approximately 70% to 33.7 s1, and the KM value increased by about 15-fold to 4.1 mM. Thus, ME-L310K/L404S bound NAD substantially weaker and retained only 2.2% catalytic efficiency of that of ME, indicating that NAD was strongly disfavored by the mutant enzyme. It should be emphasized that such a low catalytic efficiency was in fact beneficial to develop orthogonal systems depending on cofactor analogs. For ME-L310K/L404S, both kcat and KM were lower in the presence of B-2 that those in the presence of NAD. As a result, MEL310K/L404S had a very close catalytic efficiency with B-2 to that with NAD, suggesting that there was little cofactor preference between these two compounds and that B-2 did not match well with the mutant enzyme. Fortunately, ME-L310K/L404S had lower KM values of 1.1 mM and 1.0 mM, respectively, for analog B-7 and B-8, indicating that these two compounds fit much better in the engineered cofactor binding pocket. These data also suggested that ME-L310K/L404S might have a spacious cofactor binding site for NAD analogs with bulkier groups attached to the 1,2,3-triazole ring. Moreover, ME-L310K/L404S showed much higher kcat values in the presence of B-7 or B-8. As a result, catalytic efficiency of ME-L310K/L404S improved by more 10-fold to 78.8 mM1 s1 and 68.6 mM1 s1, respectively, upon switching the cofactor from B-2 to B-7 or B-8. The ratio between the kcat/KM value of ME-L310K/L404S and that of ME for each cofactor indicated the tendency of the corresponding cofactor favored by the mutant enzyme. Thus, the ratio was 0.022 in the case of NAD, indicating NAD was strongly disfavored by ME-L310K/L404S. Whereas, for these three NAD analogs, the ratios were higher than 1, indicating these analogs were favored by ME-L310K/L404S. Importantly, for enzymatic catalyzed oxidative decarboxylation of L-malate, the engineered system gained approximately 120-, 570- and 1200-fold cofactor specificity shift from NAD toward B-2, B-7 and B-8, respectively. Thus, the system ME-L310K/L404S and B-8 had the most stringent orthogonality to the natural system using ME and NAD. It should be noted that cofactor specificity for natural NAD(P)-dependent enzymes varied substantially in favoring of NAD or NADP.21–23 ME-L310K/ L404S actually had better cofactor specificity for B-8 than many natural enzymes for NAD or NADP. In summary, an activity screening between NAD analogs and ME mutants led to the identification of some mutants capable of
kcat (s
1
)
33.7 ± 1.9 16.4 ± 1.1 86.7 ± 6.6 68.6 ± 5.6
ðkcat =K M ÞMEL310K=L404S ðkcat =K M ÞME
KM (mM)
kcat/KM (mM
4.1 ± 0.42 2.5 ± 0.22 1.1 ± 0.12 1.0 ± 0.11
8.2 6.7 78.8 68.6
1
s
1
) 0.022 2.58 12.5 26.4
taking NAD analogs as the cofactor. One particular pair, MEL310K/L404S and the analog B-8 had good catalytic efficiency and cofactor specificity. Our results provided insightful information for the development of orthogonal redox system that is of particular important to precisely control engineered metabolic pathway in the biological systems.16 Acknowledgments This work was supported by the National Basic Research and Development Program of China (No. 2012CB721103) and National Natural Science Foundation of China (Nos. 21102143, 21325627). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl. 2014. 01.047. References and notes 1. Blank, L. M.; Ebert, B. E.; Buehler, K.; Bühler, B. Antioxid. Redox Signal 2010, 13, 349. 2. Pollak, N.; Dölle, C.; Ziegler, M. Biochem. J. 2007, 402, 205. 3. Ying, W. Antioxid. Redox Signal 2008, 10, 179. 4. Houtkooper, R. H.; Cantó, C.; Wanders, R. J.; Auwerx, J. Endocr. Rev. 2010, 31, 194. 5. Yang, H.; Yang, T.; Baur, J. A.; Perez, E.; Matsui, T.; Carmona, J. J.; Lamming, D. W.; Souza-Pinto, N. C.; Bohr, V. A.; Rosenzweig, A. Cell 2007, 130, 1095. 6. Holm, A. K.; Blank, L. M.; Oldiges, M.; Schmid, A.; Solem, C.; Jensen, P. R.; Vemuri, G. N. J. Biol. Chem. 2010, 285, 17498. 7. Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 651. 8. Boyce, M.; Bertozzi, C. R. Nat. Methods 2011, 8, 638. 9. Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13. 10. Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666. 11. McLachlan, M. J.; Chockalingam, K.; Lai, K. C.; Zhao, H. Angew. Chem., Int. Ed. 2009, 48, 7783. 12. Raghavan, A. S.; Hang, H. C. Drug Discovery Today 2009, 14, 178. 13. Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974. 14. Ji, D.; Wang, L.; Hou, S.; Liu, W.; Wang, J.; Wang, Q.; Zhao, Z. K. J. Am. Chem. Soc. 2011, 133, 20857. 15. Ji, D.; Wang, L.; Liu, W.; Hou, S.; Zhao, K. Z. Sci. China Chem. 2013, 56, 296. 16. Mampel, J.; Buescher, J.; Meurer, G.; Eck, J. Trends Biotechnol. 2013, 31, 52. 17. Hou, S.; Liu, W.; Ji, D.; Wang, Q.; Zhao, Z. K. Tetrahedron Lett. 2011, 52, 5855. 18. Tracewell, C. A.; Arnold, F. H. Curr. Opin. Chem. Biol. 2009, 13, 3. 19. Nobeli, I.; Favia, A. D.; Thornton, J. M. Nat. Biotechnol. 2009, 27, 157. 20. Wang, J.; Zhang, S.; Tan, H.; Zhao, Z. K. J. Microbiol. Methods 2007, 71, 225. 21. Steen, I. H.; Lien, T.; Madsen, M. S.; Birkeland, N.-K. Arch. Microbiol. 2002, 178, 297. 22. Capone, M.; Scanlon, D.; Griffin, J.; Engel, P. C. FEBS J. 2011, 278, 2460. 23. Hsieh, Y.-J.; Hung, H.-C. J. Biol. Chem. 2009, 284, 4536.