Molecular cloning, purification, and biochemical ...

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Molecular cloning, purification, and biochemical characterization of recombinant isocitrate dehydrogenase from Streptomyces coelicolor M-145 a

a

a

Tóshiko Takahashi-Iñiguez , Saul Cruz-Rabadán , Luis Miguel Burciaga-Cifuentes & María a

Elena Flores a

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, D.F., México Published online: 12 Jun 2014.

To cite this article: Tóshiko Takahashi-Iñiguez, Saul Cruz-Rabadán, Luis Miguel Burciaga-Cifuentes & María Elena Flores (2014): Molecular cloning, purification, and biochemical characterization of recombinant isocitrate dehydrogenase from Streptomyces coelicolor M-145, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.923290 To link to this article: http://dx.doi.org/10.1080/09168451.2014.923290

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Bioscience, Biotechnology, and Biochemistry, 2014

Molecular cloning, purification, and biochemical characterization of recombinant isocitrate dehydrogenase from Streptomyces coelicolor M-145 Tóshiko Takahashi-Iñiguez, Saul Cruz-Rabadán, Luis Miguel Burciaga-Cifuentes and María Elena Flores* Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, D.F., México Received November 21, 2013; accepted March 7, 2014

Downloaded by [UNAM Ciudad Universitaria] at 10:39 16 June 2014

http://dx.doi.org/10.1080/09168451.2014.923290

Isocitrate dehydrogenase is a key enzyme in carbon metabolism. In this study we demonstrated that SCO7000 of Streptomyces coelicolor M-145 codes for the isocitrate dehydrogenase. Recombinant enzyme expressed in Escherichia coli had a specific activity of 25.3 μmoles/mg/min using NADP+ and Mn2+ as a cofactor, 40-times higher than that obtained in cellfree extract. Pure IDH showed a single band with an apparent Mr of 84 KDa in SDS-PAGE, which was also recognized as His-tag protein in the Western blot. Unexpectedly, in ND-PAGE conditions showed a predominant band of ~168 KDa that corresponded to the dimeric form of ScIDH. Also, zymogram assay and analytical gel filtration reveal that dimer was the active form. Kinetic parameters were 1.38, 0.11, and 0.109 mM for isocitrate, NADP, and Mn2+, respectively. ATP, ADP, AMP, and their mixtures were the main ScIDH activity inhibitors. Zn2+, Mg2+, Ca2+, and Cu+ had inhibitory effect on enzyme activity. Key words:

Streptomyces coelicolor; dimeric isocitrate dehydrogenase; tricarboxylic acid cycle; carbon metabolism

The citric acid cycle is a central oxidative pathway in aerobic prokaryotes and eukaryotes.1) Also, it provides important biosynthetic precursors for cellular components such as α-ketoglutarate, succinyl-CoA, and oxaloacetate.2) Besides, tricarboxylic acid (TCA) cycle acts as a metabolic energy source by degradation of organic molecules like sugars and fatty acids generating acetyl-CoA via decarboxylation of pyruvate, which enters in the cycle providing reductive potential as NADH and FADH2 whose reoxidation is used for the synthesis of ATP.3) The first redox and decarboxylation steps in TCA cycle are catalyzed by a single enzyme, isocitrate dehydrogenase (IDH). It converts 2R,3S-isocitrate to 2-oxoglutarate and CO2 while reducing the coenzyme

nicotinamide adenine dinucleotide (phosphate) [NAD(P)+] to NAD(P)H.1) In prokaryotes, it has been reported both monomeric and oligomeric forms of IDH, which are phylogenetically unrelated.4) Based on coenzyme specificity, the IDH members can be classified into NAD+-dependent IDH (EC 1.1.1.41, NAD-IDH), present in eukaryotic organisms, and NADP+-dependent IDH (EC 1.1.1.42, NADP-IDH) with a ubiquitous distribution.5) NAD-IDH participates in respiratory ATP production, while NADP-IDH is involved in generating NADPH and α-ketoglutarate for biosynthesis.6) It is considered that IDH plays an important role as a carbon flow control through the citric acid cycle via negative and positive effectors.7) Streptomycetes are widely studied bacteria because they produced a large amount of hydrolytic enzymes and important compounds for medical, agricultural, and industrial applications. Thus, there are available many studies about the pathways and regulation of secondary metabolism in Streptomyces but there are relatively few studies on primary metabolism, despite being the precursor supplier for secondary metabolism.8) Within this genus, Streptomyces coelicolor A3(2) was the first strain whose genome was sequenced and it has been used as a model organism.9) Here we report that SCO7000 of S. coelicolor codes for the enzyme isocitrate dehydrogenase and the cloning and expression in Escherichia coli. The recombinant IDH was purified and its biochemical properties were determined in detail.

Materials and methods Chemicals. All chemicals were purchased at Sigma Aldrich in analytical grade. Culture conditions and media. Streptomyces coelicolor strain M145, E. coli TOP10F′ (Invitrogen), and E. coli BL21(DE3)pLysS (Invitrogen) were used in this

*Correspondence author. Email: mfl[email protected] Abbreviations: BSA, Bovine seric albumin; IDH, Isocitrate dehydrogenase; LB, Luria Bertani; ScIDH, Streptomyces coelicolor Isocitrate dehydrogenase; TCA, Tricarboxylic acid. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry


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T. Takahashi-Iñiguez et al.

Fig. 1. Purification, identification, gel filtration, and zymogram assay of recombinant ScIDH. Notes: (A) SDS-polyacrylamide gel (8%) stained with Coomassie Brilliant Blue G-250, M, molecular mass marker; Lane 1, purified IDH (7 μg). (B) Detection of His-tagged IDH by Western blot, Lane 1, purified IDH (10 μg); M, molecular mass marker. The migration position of IDH is indicated by an arrow (~84 KDa). (C) ND-polyacrylamide gel (9%) stained with Coomassie Brilliant Blue G-250, M, molecular mass marker; Lane 1, purified IDH (10 μg); Lane 2, Filtration profile on a Macro-Prep SE 1000/40 gel column (1.2 mg of purified IDH); Lane 3, Zymogram assay of the individual protein peaks.

work. In general, S. coelicolor was maintained as spore suspension in 20% glycerol and kept at -20 °C until use. It was grown on liquid or solid YEMEG medium10) at 29 °C and 200 rpm when needed. Luria–Bertani’s medium with appropriate antibiotics was used for E. coli. Cloning of idh gene. Based on the sequence of chromosomal DNA of S. coelicolor reported (SCO7000), oligonucleotides were designed to use in PCR amplification of putative idh gene. Forward and reverse oligonucleotide sequence were 5′-AGCGGAGCTCGAGATGGACTGACTCGA-3′ and 5′GCGGGGAATTCGAGGATCAGG-3′, respectively, including unique XhoI and EcoRI restriction sites for cloning in pRSETA (Invitrogen). The obtained amplicon of 2220 nt and pRSETA plasmid were purified from the agarose gel by using QIAquick Gel Extraction Kit (Qiagen). The DNA fragment was ligated into the XhoI and EcoRI sites of the expression vector. The resulting construct was pRSETA-SCidh. Expression and purification of IDH. For expression of recombinant IDH, chemically competent cells of E. coli BL21(DE3)pLyS were transformed with pRSETA-SCidh and selected in LB broth with ampicillin (100 μg mL−1) and chloramphenicol (35 μg mL−1) plates. One transformed clone was used to inoculate LB medium with ampicillin (100 μg mL−1) and chloramphenicol (35 μg mL−1). E. coli was grown for ~6 h at 22 °C with shaking (200 rpm); when the culture reached an OD600 between 0.4 and 0.6, protein expression was induced with 0.5 mM IPTG (Sigma) for 2 h (200 rpm). The expressed His-tag protein was purified by using Dynabeads magnetic beads His-Tag Isolation and Pulldown kit following the manufacturer’s instructions (Novex). Purity and western blot analysis. The protein purity was verified by SDS-PAGE. His-tagged IDH was identified in western blot using the anti-HisG-AP antibody (Invitrogen) and detected with the BCIP/NBT substrate kit (Invitrogen) according to the manufacturer’s instructions.

Zymogram. Zymogram was done by electrophoresis of IDH in non-denaturing conditions using NADP+ (0.5 mM) in run buffer. After run, gel was rinsed twice in universal buffer (Boric acid 27.5 mM, citric acid 28.5 mM, and KH2PO4 28 mM; pH 7.8) and the activity was developed by incubation at 22 °C in a reaction mixture containing isocitrate (30 mM) and MnSO4 (5 mM) in universal buffer (Boric acid 27.5 mM, citric acid 28.5 mM, KH2PO4 28 mM; pH 7.8). IDH activity was detected as UV absorbing bands.11) Size exclusion chromatography. The separation of trimeric, dimeric, and monomeric forms of IDH was carried out by analytical gel filtration on a column of 50 × 1 cm with Macro-Prep SE1000/40 gel (Bio-Rad) equilibrated with universal buffer (pH 7.8). The Bio-Rad size exclusion standards were used for column calibration. Enzyme assay and kinetic data. NADP+-Isocitrate dehydrogenase was assayed by the method reported by Wang et al.12) The standard assay was performed at 22 °C in a reaction mixture containing NADP+ (0.5 mM), isocitrate (15 mM), and MnSO4 (2 mM) in universal buffer (pH 7.8). Kinetic parameters were determined by varying the concentrations of NADP+ (0.001–1 mM), isocitrate (0.25–60 mM), and MnSO4 (0.005–8 mM) in independent assays maintaining the concentration of the invariable substrates as described for the standard assay. All kinetic parameters were obtained from at least three independent measurements. Protein concentration was determined by the Bradford protein assay using BSA as a standard.13) Optimum temperature and pH determination. The optimum temperature was determined by measuring enzyme activity under the same conditions described for standard assay at distinct temperatures (30–60 °C). To determine the optimum pH of the enzyme, activity was assayed in universal buffer between pH 7 and 11; potassium phosphate buffer (0.1 M) was used for values of pH between 5 and 8.

Streptomyces coelicolor isocitrate dehydrogenase

Different compounds and metal ion effects. The activity of recombinant IDH from Streptomyces coelicolor (ScIDH) was measured in the presence of various compounds (ATP, ADP, AMP, GTP, GDP, citrate, α-ketoglutarate, oxaloacetate, and glyoxalate) and combinations thereof at 5 mM under the same standard assay conditions. The effect of metal ions was determined by measuring IDH activity in presence of 2 mM of monovalent ions (K+ and Na+) and divalent ions (Zn2+, Mg2+, Co2+, Cu2+, and Ca2+) instead and besides MnSO4 under the same standard assay conditions.


Results and discussion Cloning, expression, purification, and western blot assay of ScIDH To identify the gene encoding ScIDH, analysis of the complete genome of S. coelicolor was done resulting SCO7000 ORF as probable isocitrate dehydrogenase and predicted a monomeric IDH.14) BLAST analysis using the amino acid sequence deduced from the nucleotide sequence of SCO7000 showed that this protein has 99% identity with IDH from Streptomyces lividans TK54 (EU661252), 91% with IDH from Streptomyces diastaticus (ADC36213.1), 88% with IDH from Streptomyces avermitilis (NP_828390.1), and 68% with IDH from Corynebacterium glutamicum (WP_011013800.1). By using primers designed, an expected amplicon of 2220 bp was obtained using DNA from S. coelicolor M-145 as a template. It was cloned into pRSETA expression vector in the corresponding restriction sites and the resulting pRSETA-SCidh vector was transformed in E. coli for enzyme expression. The expression at 37 °C resulted in a large amount of inclusion bodies so the temperature was decreased to 22 °C obtaining a higher yield of soluble IDH. It is well-known that lowering temperature of E. coli cultivation increased the amount of soluble recombinant protein produced.15) Recombinant protein ScIDH was purified by affinity using magnetic beads and 10 mg of pure protein per liter of culture medium was obtained. In SDS-PAGE, a single band with an apparent Mr of 84 KDa was found for pure IDH protein (Fig. 1(A)), which was also recognized as His-tag protein in the Western blot (Fig. 1(B)). The molecular weight obtained is consistent to the expected taking into account the addition of His-tag.

IDH activity and Zymogram As described in other actinomycetes6,16), S. coelicolor IDH was NADP+ and Mn2+/Mg2+ dependent and had a maximum activity of 25.3 μmoles/mg/min. Unexpectedly, in polyacrylamide gel under native conditions (Fig. 1(C)) showed a predominant band of ~168 KDa that corresponded to the homodimeric form and a ~252 KDa protein band that could be a trimer of ScIDH. Monomer protein band was not visualized, probably due to a very low concentration. Dimer and trimer were resolved by analytical gel filtration but the monomer peak could not be detected. The dimeric protein peak showed a specific activity of 28 μmoles mg−1 min−1 and zymogram assay also revealed that dimer form was active, while the trimeric form had no activity (Fig. 1(C)).

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Despite the fact that ScIDH had high identity (between 80 and 90%) and similar molecular masses (between 70 and 79 KDa) with S. lividans TK546) S. diastaticus,16) and S. avermitilis,12) their enzymes have been described as monomers, contrary to that observed in S. coelicolor. Effects of pH and temperature on ScIDH activity To determinate the optimal activity pH, two different buffers were required. To cover the pH range from 5 to 7.5 phosphate buffer was used; for pH range from 7 to 10 universal buffer was utilized. In both cases the maximum ScIDH activity was obtained around 7.5, having an optimal value in pH 7.8 in universal buffer. Also, a drastic decrease in activity was observed at basic pH values. These data are consistent with the ranges reported for dimeric NADP-IDHs as E. coli17) (pH 7– 9), Thermus thermophilus18) (pH 7.4), Bacillus sp.19) (pH 7.8–8.4), and S. erythraea7) (pH 7.5–9.5) (Fig. 2). With respect to the optimum temperature, our results showed a maximal activity at 50 °C falling rapidly at higher temperatures (Fig. 3). Several IDHs with optimal temperature around 50 °C have been reported, such as from Azotobacter vinelandii20) (40–50 °C), S. lividans6) (46 °C), Rhodomicrobium vannielii21) (50 °C), and Desulfobacter vibrioformis (45 °C)22). Kinetics analysis Km and Vmax determinations for substrate and cofactors were done by measured activity in presence of different concentrations of isocitric acid, NADP+, and Mn2+ at 25 °C. For the three compounds, the enzyme showed a Michaelis–Menten kinetics and results in Table 1 shown that ScIDH recombinant enzyme had low affinity for NADP+. ScIDH Km value for NADP+ (0.11 mM) was similar to the value obtained for Helicobacter pylori IDH.23) In general, it has been reported that monomeric IDHs have more affinity for NADP+ than dimeric forms.6) Km values for isocitrate and Mn2+ are also high and were not similar to none previously reported. No activity was found with NAD+ as a cofactor (data not shown).

Fig. 2. Optimal pH profile. The optimal pH of IDH was determined in universal (○) or phosphate buffer (●) depending on the pH range.

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T. Takahashi-Iñiguez et al. Table 2. Effect of different compounds on the activity of recombinant IDH.a Compound (5 mM)


Fig. 3. Optimal temperature profile. Notes: The optimal temperature of recombinant IDH was determinate by incubation of the enzyme from 30 to 60 °C under standard assay conditions described in materials and methods.

Table 1.

Kinetic parameters of recombinant ScIDH. a

Substrate

Km (mM)

Isocitrate NADP Mn2+

1.38 0.11 0.109

a

−1

Vmax (μmol min

−1 a

mg )

25.31 26.31 27.39

The values indicate the means of at least three independent measurements.

Positive and negative effectors of IDH activity In biological systems, metabolic regulation of catabolic pathways is carried out by enzyme inhibition. Intermediaries of TCA cycle typically act as inhibitory molecules of IDHs activity in various organisms. An example of that is the IDH from S. erythraea; molecules like ATP, citrate, oxaloacetate, or α-ketoglutarate are negative effectors of its activity.7) As can be seen in Table 2, ATP, ADP, and AMP at 5 mM concentration inhibited the activity between 30 and 60% under the conditions tested. The guanosine nucleosides inhibit 30% approximately. This data make sense because the function of the TCA is to generate energy in the form of ATP. An excess of these molecules acts as an indicator of the presence of high energy signal decreasing the TCA flux in consequence. To assess the possibility of a synergistic effect, mixtures of the different nucleosides were made. The maximum inhibition was obtained in the mixture containing the three adenine nucleosides (80%). Similar inhibition was reported for NADP+-IDH from S. erythraea.7) Citrate, α-ketoglutarate, oxaloacetate, glyoxalate, and their combinations added to the activity assay compounds had no inhibitory effect; on the contrary, a slight increase in activity (7–10%) was observed when they were present. Effect of various metal ions on the activity of the ScIDH The enzyme activity was determined in presence of various mono and divalent metal ions, all of them at a final concentration of 2 mM (Table 3). No activity was detected in absence of divalent ions, which demonstrates that ScIDH activity was totally dependent on the binding of cations. The maximum activity of ScIDH

None ATP ADP AMP ATP+ADP ATP+AMP ADP+AMP ATP+ADP+AMP GTP GDP GTP+GDP Citrate α-Ketoglutarate Oxaloacetate Glyoxalate α-Ketolgutarate + Oxaloacetate α-Ketolgutarate + Glyoxalate α-Ketolgutarate + Oxaloacetate + Glyoxalate Citrate + α-Ketolgutarate + Oxaloacetate + Glyoxalate

Activity (μmol min−1 mg−1)

Relative activity (%)

29.26 11.70 15.57 20.29 7.54 9.61 13.55 6.87 18.85 20.64 12.89 29.13 32.52 31.15 32.73 31.59

100 ± 4.56 39.99 ± 1.87 53.20 ± 2.13 69.34 ± 0.35 25.79 ± 1.63 32.85 ± 2.73 46.30 ± 1.86 23.50 ± 2.20 64.43 ± 3.83 70.54 ± 0.90 44.07 ± 3.88 99.54 ± 3.93 111.12 ± 0.59 106.44 ± 2.75 111.85 ± 1.67 107.95 ± 1.49

31.41

107.34 ± 1.15

27.27

93.194 ± 2.01

31.59

107.95 ± 1.49

a


was observed when assayed with Mn2+ followed by Zn2+ and Mg2+. The preference for Mn2+ has been observed in other actinomycetes like S. lividans,6) S. diastaticus,16) and S. avermitilis.12) No activity was observed in presence of Ca2+, Cu2+, K+, and Na+. On

Table 3. IDH.a Metal ion None Mn2+ Zn2+ Mg2+ Co2+ Cu2+ Ca2+ K+ Na+ Mn2+ Mn2++Zn2+ Mn2++Mg2+ Mn2++Co2+ Mn2++Cu2+ Mn2++Ca2+ Mn2++K+ Mn2++Na+ a

Effects of metal ions on the activity of recombinant

Relative activity (%) 0 100.00 ± 5.53 24.17 ± 0.93 20.01 ± 1.01 18.77 ± 3.39 4.02 ± 0.92 0.86 ± 0.58 3.92 ± 1.05 3.34 ± 1.89 100.00 ± 5.53 88.32 ± 1.77 79.30 ± 4.23 112.00 ± 1.72 94.73 ± 3.16 73.31 ± 2.15 105.09 ± 2.75 100.9 ± 0.44


Streptomyces coelicolor isocitrate dehydrogenase 2+

2+

2+

2+

the other hand, Zn , Mg , Ca , and Cu when added besides Mn2+ had a slightly inhibitory effect on IDH activity. The results reported here showed that SCO7000 codes for S. coelicolor isocitrate dehydrogenase, not a monomeric but dimeric enzyme with a low affinity for the substrates. Nonetheless, the activity inhibition by metabolites indicates that IDH continues to be a key enzyme in the regulation of the TCA cycle in this micro-organism. Finally, it is important to mention that we could not obtain an icdh- mutant (data not shown) probably because it is an indispensable protein confirming that IDH is a crucial enzyme in the TCA cycle to maintain carbon flux and generate precursors for secondary metabolism.


Funding This work was supported by a CONACyT, México [grant number 58060].

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