Escherichia coli Strain Designed for Characterizing in Vivo Functions

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Escherichia coli Strain Designed for Characterizing in Vivo Functions of Nicotinamide Adenine Dinucleotide Analogues Lei Wang,† Bin Liu,† Yuxue Liu,‡ Yue Sun,† Wujun Liu,§ Dayu Yu,*,† and Zongbao K. Zhao*,‡ †

School of Chemical Engineering, Northeast Electric Power University, Jilin 132012, China Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China § Institute of Cancer Stem Cell, Dalian Medical University, Dalian 116044, China ‡

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ABSTRACT: An Escherichia coli strain was constructed for the efficient import of nicotinamide adenine dinucleotide (NAD) analogues into cells by limiting extracellular degradation while expressing an efficient NAD importer. In vivo functions of three NAD analogues were characterized. Nicotinamide hypoxanthine dinucleotide was identified as an inhibitor of NAD synthesis. Nicotinamide cytosine dinucleotide had excellent biocompatibility and was used for characterizing a growth-dependent degradation of in vivo nicotinamide cofactors.

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main pyrophosphatase for the decomposition of exogenous cofactors. Then, nicotinamide cofactors can be directly, continuously, and efficiently imported. Three NAD analogues (NXDs, Figure 1) were designed and synthesized to test the substrate spectrum and activity of the importing system.8 In vivo functions of the cofactors were characterized. The NXDs were designed with the adenine group of NAD substituted by natural bases. NXD with a natural base might have excellent biocompatibility and be synthesized via modified endogenetic NAD salvage pathways,9 which will facilitate in vivo applications after being characterized. Based on the structure similarity, the newly reported nicotinamide hypoxanthine dinucleotide (NHD, 1a) with a purine group might have similar character with NAD and possess different properties toward nicotinamide cytosine dinucleotide (NCD, 2a) and nicotinamide uracil dinucleotide (NUD, 3a) with a pyrimidine group. The intracellular cofactors were analyzed by enzymatic cycling assay. The assay amplified the signal by cycling cofactors via transferring reduction power to oxidized thiazolyl blue tetrazolium bromide (MTTOX) using phenazine ethosulfate (PES), and the reaction was monitored by absorbance of reduced MTT (MTTRE) at 570 nm (Table 1).5 As the cofactor structures have distinct influences on the Km and activity of redox enzymes (REs),4,5 the applicability of the cofactor analysis by enzymatic cycling assay with alcohol dehydrogenase from Saccharomyces cerevisiae (Adh) and mutant malic enzyme from Escherichia coli (Mae*) needs confirmation. The cofactor concentration extracted from the cell was no more than 40 μM,10,11 and the influence of cofactor

icotinamide adenine dinucleotide (NAD, Figure 1) and its phosphorylated form (NADP) play a central role as a

Figure 1. Structure of NAD and NAD analogues with the adenine group substituted by natural bases.

nicotinamide cofactor in biology.1 However, efforts to study the roles of NAD(P) have proven difficult because these cofactors are subjected to tight regulation and participate in complex metabolic networks. NAD analogues can enhance our ability to understand the mechanism underlying bioenergetic and signaling pathways mediated by NAD and provide effective strategies for the regulation of redox reactions.2−4 However, little in vivo application of NAD analogues has been reported,5 and their application was limited to in vitro use2,6,7 because these analogues cannot be easily delivered into cells. We envisaged cofactor transporters (CTs) as a general means for importing cofactors; sufficient stability of the cofactors in culture media is required. Here, we optimized the importing efficiency of nicotinamide cofactors by choosing an efficient transporter and deleting the gene encoding the © XXXX American Chemical Society

Received: March 15, 2019

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DOI: 10.1021/acs.orglett.9b00935 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters Table 1. Relative Activity of Adh and Mae* with Different Cofactorsa

Table 2. Importing Efficiency of NTT4 and AtNDT2 for NAD and NCDa

entry

entry

REsb

cofactor

relative activityc

1 2 3 4 5 6 7 8

Adh

NAD NHD NCD NUD NAD NHD NCD NUD

100 7.9 0.94 1.2 1.5 1.5 100 14

Mae*

1 2 3 4

CTsb NTT4 AtNDT2

cofactor

importing rate (mM/h)c

NAD NCD NAD NCD

0.23 0.15 0.066 0.013

a

Conditions: Cells were incubated in the presence of 0.1 mM cofactor at 30 °C for 8 h. bNTT4: CT from P. amoebophila UWE25.11 AtNDT2: CT from A. thaliana.5 cValues are expressed as increased intracellular concentrations of NAD or NCD per hour.

0.1 mM NCD. The subsequent import was conducted by NTT4. Transport via NTT4 requires suppressing the decomposition of cofactors mediated by phosphatases. To limit or compensate extracellular decomposition of the cofactors, cofactors are generally imported by decreasing operating time14−16 or supplying excess cofactors to the cultural medium.11,15 In E. coli, the extracellular pyrophosphatase UshA hydrolyzes the nicotinamide cofactors to permeable nicotinamide riboside (NR) and nucleosides.17 We expected that deletion of the ushA gene should significantly increase the stability of cofactors in culture media (Figure 2A).8 The Km values for NAD of UshA and NTT4 were 5.3 μM and 15 μM, respectively,14,17 so we expected 50 μM exogenously added

Conditions: [substrate]0 = 5 mM, [cofactor]0 = 50 μM, [Adh] = 1 U/mL, [Mae] = 1.7 U/mL, [MTTOX]0 = 0.4 mM, [PES]0 = 1 mM, T = 30 °C, reaction time: 10 min. bAdh: RE specific to NAD.11 Mae*: RE specific to NCD.5 cValues are expressed as percent relative activity compared to preferred cofactor, which is assigned a value of 100. a

structure on the activity with assay REs was investigated with 50 μM cofactors (Table 1). Though NHD had a similar structure to NAD, the NAD-specific Adh retained only 7.9% activity with NHD compared with NAD (entries 1 and 2). For NCD-specific Mae*, the activity with NUD was 14% of the activity with NCD (entries 7 and 8). NCD and NUD were poorly used by Adh, whereas NAD and NHD were poorly used by Mae* (entries 3−6). The cofactor structure has a critical influence on enzyme activity, and only NAD and NCD could be analyzed using the enzymatic cycling assay. NAD and NCD were used for the design and characterization of an efficient NXD importing system. The efficient CT with wide substrate spectrum was chosen between AtNDT2 derived from Arabidopsis thaliana and NTT4 derived from Protochlamydia amoebophila UWE25. Among the well-characterized CTs, AtNDT2 has the widest substrate spectrum and the highest activity.12,13 AtNDT2 has been applied for importing NCD for construction of in vivo metabolic circuits.5 To date, NTT4 has been proven to be an excellent transporter for NAD, but its substrate spectrum has not been well studied.11,14 The cofactor transporting efficiencies of NTT4 and AtNDT2 were compared (Table 2).8 Compared with AtNDT2, NTT4 was a more efficient transporter for both NAD and NCD (entries 1−4). With 0.1 mM exogenously added cofactors, NTT4 increased the cellular NAD and NCD level by 1.8 mM and 1.2 mM, respectively, in 8 h. During the same time course, AtNDT2 only increased the cellular NAD and NCD levels by 0.5 mM and 0.1 mM, respectively. Both NTT4 and AtNDT2 preferred NAD to NCD. The importing efficiency of NTT4 was 3−16-fold greater than AtNDT2. NTT4 could concentrate exogenous cofactor into cells via counter exchange with ADP14 and created a 12-fold higher intracellular NCD concentration than the exogenously added

Figure 2. Reducing extracellular cofactor degradation. (A) Degradation of cofactors by UshA. (B, C) Time course of cofactor degradation by wild-type (WT) and ushA-deletion mutant (ΔushA) E. coli. (D, E) Time course of cofactor import by WT and ΔushA E. coli. Cells were incubated in the presence of 50 μM cofactor at 30 °C. The data represent the average standard deviations of three independent experiments. B

DOI: 10.1021/acs.orglett.9b00935 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

data, NTT4 has broad affinity for NXDs and prefers NXDs with a purine group to NXDs with a pyrimidine group. Import of NXD has different influences on intracellular NAD compared to the control without exogenously added cofactors (Figure 3A).8 The impact of NXD on intracellular NAD concentration was assayed using strains with and without NTT4 (Figure 3B). When supplemented with 0.5 mM NXD, little impact on the intracellular NAD concentration of cells without NTT4 was detected. When supplemented cells express NTT4 with 0.5 mM of NHD, the intracellular NAD concentration was lower than the sample without NHD (Figure 4B). This suggests that NHD potently inhibits

cofactors to be sufficient for investigating the degradation and import process. When NAD and NCD were incubated with ushA deletion mutant for 12 h, the cofactors remained stable, and the residual NAD and NCD concentrations remained at 37 μM and 23 μM, respectively (Figure 2B, C). When incubated with wild-type strain cells, NAD and NCD were almost completely degraded within 6 and 4 h, respectively (Figure 2B, C). According to the degradation rate of the cofactors in the initial 2 h, deletion of the ushA gene improved extracellular NAD and NCD stability by 8- and 11-fold, respectively. NAD was more stable than NCD in both cells with or without UshA. Corresponding to extracellular NAD and NCD degradation, detectable import of NAD and NCD by wild-type cells stopped at 5 and 2 h (Figure 2D,E),8 respectively. For NAD, wild-type cells maximally increased the intracellular NAD concentration by 1.4 mM at 6 h, whereas ushA-deletion mutant cells increased intracellular NAD concentration by 2.8 mM at the same time. The concentration reached 3.3 mM at 12 h. For NCD, wild-type cells maximally increased intracellular NCD concentration by 0.34 mM at 4 h, whereas ushA-deletion mutant cells increased intracellular NCD concentration by 0.95 mM at the same time. The concentration reached 2.0 mM at 12 h. That is, cells with ushA-deletion mutant could continuously and efficiently import NAD and NCD. The efficient cofactor-importing strain might also be able to import other NXDs with purine or pyrimidine groups. The affinity of NXDs by NTT4 was characterized by inhibition of the uptake of NAD (Figure 3A).14,18 The cells were incubated with 50 μM NAD and a 10-fold excess in NXD. NHD potently inhibited NAD uptake (73.4% inhibition), whereas NCD and NUD inhibited uptake much less efficiently (40.6% and 53.6% inhibition, respectively). Based on this competitive inhibition

Figure 4. Intracellular cofactor concentration before and after incubation in M9 medium with and without glucose. (A) Time course of intracellular NCD degradation by wild-type and ushAdeletion mutant E. coli. (B) Time course of intracellular NAD concentration in wild-type and ushA-deletion mutant E. coli. Cells were collected after 12 h incubation with 0.1 mM NCD and resuspended in M9 medium with or without 2% glucose. Then the cells were incubated at 30 °C for 6 h. The data represent the average standard deviations of three independent experiments.

intracellular NAD synthesis. Intact NHD was necessary for the inhibition of NAD synthesis, and its degraded products (inosine and NR) did not inhibit NAD synthesis (Figure 3C). When 0.5 mM NXD was supplemented to cells with NTT4, NCD and NUD increased intracellular NAD concentration by approximately 42%. The increase in intracellular NAD concentration might be due to NR or nucleosides produced by the intracellular degradation of NXD. This hypothesis was confirmed by the impact of NXD precursors on intracellular NAD concentration (Figure 3C). NR, nicotinamide, and nicotinic acid stimulated the synthesis of NAD and increased intracellular NAD concentration approximately by 1-fold, which is consistent with the phenomenon in mice and humans.19 Nucleotide precursors had little impact on NAD metabolism. None of the NR precursors or nucleotide precursors had an impact on NAD uptake (Figure 3C). Besides regulating the metabolism of NAD, NAD analogues can serve as functional and mechanistic in vivo probes.20,21 As natural cofactors are subjected to tight regulation and participate in complex metabolic networks, it is difficult to investigate the maintenance of NAD pools. NCD can serve as an indicator of intracellular degradation activity because NCD cannot be synthesized by E. coli and has an excellent biocompatibility with little influence on cell metabolism (Figure S1).5 In rest cells, intracellular NCD decreased from 0.34 mM at 4 h to 0.30 mM at 12 h, which suggests cofactors are more stable in the rest cell cytoplasm than in the extracellular environment (Figure 2E). For growing cells, the growth status (Figure S2) and the intracellular cofactor stability (Figure 4) were investigated by incubating the cells preloaded with NCD in M9 medium with glucose. The cells

Figure 3. Effects of NXD on NAD import. (A) Inhibition of uptake of NAD by the NXDs. Cells were mixed with 500 μM NXD and 0 or 50 μM NAD. (B) Influence of NXD import on intracellular NAD concentration. E. coli with or without NTT4 was mixed with 0.5 mM NXD. (C) Impact of NXD precursors on intracellular NAD concentration and uptake of NAD. WL023 cells were mixed with 500 μM precursors and 0 or 50 μM NAD. The data represent the average standard deviations of three independent experiments. C

DOI: 10.1021/acs.orglett.9b00935 Org. Lett. XXXX, XXX, XXX−XXX

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incubated without glucose stopped growing after 2 h, whereas cells incubated with glucose kept growing during the process (Figure S2). Accompanied by growth status, intracellular NCD in wild-type cells decreased from 1.7 mM to 1.0 mM and 0.65 mM after incubation with and without glucose for 6 h, respectively. For ushA-deletion mutant cells, intracellular NCD decreased from 1.9 mM to 1.3 mM and 0.71 mM after incubation with and without glucose, respectively (Figure 4A). Whereas intracellular NCD decreased, intracellular NAD increased during the incubation (Figure 4B), and this increase was also observed in rest cells (Figure S3). This suggests that the intracellular cofactor degradation is growth dependent, and the degradation in cells incubated without glucose might be due to the growth during the initial period of resuspension. Overall, we realized the continuous and efficient import of NXDs in a designed E. coli strain. The potential application of NXDs was evaluated in vivo. NHD was identified as an inhibitor of NAD synthesis with potential application in NAD metabolism regulation and anticancer chemotherapy.22 NCD was proven to be an excellent bioorthogonal cofactor with good biocompatibility and was used for characterizing a growth-dependent in vivo degradation of nicotinamide cofactors. Using the efficient cofactor importing system, the bioorthogonal analogues for energy transmission,4 cheap synthetic analogues with high activity,2,6 and analogue server as a probe of NAD-dependent modification of biomacromolecules7,20 can be used in the cellular environment. This will facilitate the application of cofactor analogues.

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REFERENCES

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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.9b00935. Experimental procedures and full characterization and spectroscopic data for compounds (1−3)a and strains (PDF)

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Letter

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.Y.) *E-mail: [email protected] (Z.K.Z.) ORCID

Dayu Yu: 0000-0001-7082-6762 Author Contributions

L.W. and Z.K.Z. conceived and designed the experiments. L.W., B.L., and Y.S. performed the experiments. L.W., Y.L., W.L., and D.Y. analyzed data. L.W., D.Y., and Z.K.Z. wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank H. Ekkehard Neuhaus (Technische Universität Kaiserslautern, Germany) and Ferdinando Palmieri (Università degli Studi di Bari Aldo Moro, Italy) for providing AtNDT2. This study was funded by the National Natural Science Foundation of China (grant number 21708003, 31470787, 21837002) and Science and Technology Research Project of Jilin Province, China (grant number 20170519015JH). D

DOI: 10.1021/acs.orglett.9b00935 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters degradation in Escherichia coli. Enzyme Microb. Technol. 2014, 58−59, 75−79. (18) Ast, M.; Gruber, A.; Schmitzesser, S.; Neuhaus, H. E.; Kroth, P. G.; Horn, M.; Haferkamp, I. Diatom plastids depend on nucleotide import from the cytosol. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 3621−3626. (19) Trammell, S. A.; Schmidt, M. S.; Weidemann, B. J.; Redpath, P.; Jaksch, F.; Dellinger, R. W.; Li, Z.; Abel, E. D.; Migaud, M. E.; Brenner, C. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Nat. Commun. 2016, 7, 12948. (20) Halle, F.; Fin, A.; Rovira, A. R.; Tor, Y. Emissive synthetic cofactors: enzymatic interconversions of tzA analogues of ATP, NAD+, NADH, NADP+, and NADPH. Angew. Chem., Int. Ed. 2018, 57, 1087−1090. (21) Buhrmann, C.; Shayan, P.; et al. Sirt1 is required for resveratrolmediated chemopreventive effects in colorectal cancer cells. Nutrients 2016, 8, 145−165. (22) Hasmann, M.; Schemainda, I. FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis. Cancer Res. 2003, 63, 7436−7442.

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DOI: 10.1021/acs.orglett.9b00935 Org. Lett. XXXX, XXX, XXX−XXX