Identification and Characterization of Protein Encoded by orf382 as L-Threonine Dehydrogenase
Identification and Characterization of Protein Encoded by orf382 as L-Threonine Dehydrogenase
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 748-755
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : December 11, 2013
  • Accepted : March 18, 2014
  • Published : June 30, 2014
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Fei Ma
Tianwen Wang
Xingyuan Ma
Ping Wang

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant 6×His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates ( n -butanol, L -threonine, ethanol, isopropanol, glucose, glycerol, L -serine, lactic acid, citric acid, methanol, or D -threonine), the enzyme showed the highest activity on L -threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of L -threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an L -threonine dehydrogenase (LTDH). When using L -threonine as the substrate, the enzyme exhibited the best catalytic performance at 39℃ and pH 9.8 with NAD + as the cofactor. The determination of the Km values towards L -threonine (Km = 11.29 μM), ethanol (222.5 μM), and n -butanol (8.02 μM) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the α-helix percentage (from 12.6% to 6.3%).
Advances in DNA sequencing technologies are now making it much easier to access and determine the genome of any species in the world [19] . As a result, the sequence data being deposited in international databases is increasing at an incredibly high rate [2] . In addition, developments in metagenomics, which mainly deals with mixed DNA samples from different environments, are also producing extensive sequence data [26] . One compulsory operation when processing such sequence data is to make an electronic inference of their putative function(s) by comparing the newly obtained sequences with database sequences that already have known functions [16 , 30] . This process is called genome annotation. Yet, although a powerful process, genome annotation has certain recognized limits: (i) it is impossible to make an inference for each open reading frame (orf) when using a knowledge database as a reference, and (ii) the relationship between the function of a protein and its amino acid sequence is too complicated to generalize its conserved motif for a group of proteins with a known function. Therefore, errors are inevitable. For example, in the study by Moon et al. [20] , they confirmed that the gene named invF was misannotated owing to a failure in identifying the correct starting codon. Thus, despite the normal robustness of genome annotation, experimental investigation of the biochemical properties of an unknown protein is also important, even for a protein from perhaps the most extensively studied strain E. coli . In the E. coli genome, orf382 has been annotated to encode an alcohol dehydrogenase (ADH), downstream of which is orf542 that has been identified to encode an aldehyde dehydrogenase (AlDH). When working together, these two enzymes complete the oxidation of an alcohol into an acid [31] . In the current study, the orf382 in the E. coli MG1655 genome was experimentally confirmed as an L -threonine dehydrogenase (LTDH), which is significantly beyond the simple annotation of an ADH [8 , 25] .
As reported in the study by Newman et al. [21] , LTDH is involved in the catabolism of threonine and the synthesis of glycine in E. coli K-12. They also showed that LTDH in E. coli K-12 functions in the first step in the threonine degradation pathway via the formation of α-amino-β-ketobuyrate (AKB) [24] to produce glycine. In their studies, the cell extracts had an average specific activity of 0.0186 U/mg (during the formation of the aminoketone) and an optimal pH above pH 9. In another early study, it was reported that the extracts of E. coli B/2 could be used as an NA D -linked enzyme with an optimal pH of 9.6 [28] . However, none of these studies identified the gene encoding the enzyme, focusing more on the metabolic role of the enzyme in E. coli .
Accordingly, this study cloned the orf382 from the E. coli MG1655 genome based on the gene sequence from NCBI [25] . After overexpressing the gene in E. coli BL21(DE3), the recombinant 6×His-tag protein was purified using an Ni- NTA matrix. Various biophysical and biochemical parameters of the protein were then determined. Meanwhile, a mutant strain with a disrupted orf382 (also a glycine auxotroph) was constructed to identify its function in vivo . Based on the results, the protein encoded by orf382 was confirmed to be an LTDH, as it catalyzed the catabolism of threonine into glycine. This study also showed the necessity of experimentally characterizing each protein, even in the case of proteins from the most common species E. coli [1 , 20] .
Materials and Methods
- Chemicals
The isopropyl β- D -1-thiogalactopyranoside (IPTG) and kanamycin were obtained from Sigma-Aldrich. The Ni-NTA matrix used to purify the 6×His-tag protein was a product of Biorad. The NAD + , NADP + , NADH, L -threonine, D -threonine, L -serine, Tris, and imidazole were all purchased from Sangon (Shanghai, China). The methanol, ethanol, n -butanol, glucose, isopropanol, glycerol, lactic acid, and citric acid were all from Fisher Scientific. The other chemicals used for the assays were commercially available products of analytical grade from local providers. All the solutions were made up with MilliQ water.
- Bacterial Strains, Plasmids, and Culture Medium
The genomic DNA from a lab stock of E. coli MG1655 was extracted using a kit (Transgen, Shanghai) and then used as the template for amplifying the orf382. E. coli DH5α from Invitrogen and E. coli BL21(DE3) from Novagen were used for the plasmid propagation and protein overexpression, respectively. The mutant E. coli MG1655 carrying the disrupted orf382 was constructed based on RED recombination by RUIDIBIO (Shanghai, China). The components for the polymerase chain reaction (PCR), restriction enzymes ( Nhe I and Xho I), and T4 DNA ligase were all products of Takara (Dalian, China). The kits for the plasmid extraction, PCR product cleaning, and DNA recovery from the gel after electrophoresis were purchased from Sangon. The oligonucleotide as a primer was ordered from BGI, and the pET-28a(+) for cloning and expression was a product of Novagen. The LB medium and plate (LB medium with 2% agar) were prepared using yeast extract (5 g/l), peptone (10 g/l) (Oxide, UK), and NaCl (10 g/l). The antibiotic kanamycin was supplemented to a final concentration of 50 mg/l.
- Molecular Cloning, Protein Expression, Purification, and Enzymatic Activity Assays
The encoding region from the genomic DNA of E. coli MG1655 was amplified by PCR on an ABI thermocycler using two primers specifically designed for the open reading frame (orf382): P1 (5’-CTA GCTAGC ATGGCAGCTTCAACGTTCTTTATTC-3’, underlined part is the restriction site for Nhe I) and P2 (5’-CCG CTCGAG TTA CCTCGCTGCA TAAATCGCCACA-3’, Xho I). The PCR mixture contained (50 μl) genomic DNA 1 μl, P1 and P2 2 μl each, dNTP (10 mM) 2 μl, 10×Buffer for ExTaq polymerase (Takara) 5 μl, ExTaq polymerase 1 μl, and MilliQ water 37 μl. The thermocycling parameters were set as follows: 95℃ for 3 min, 95℃ for 30 sec, 55℃ for 20 sec, and 72℃ for 70 sec for 30 cycles, followed by an extension for 7 min. After confirming the successful amplification with agarose electrophoresis, the PCR product and plasmid pET28a(+) were digested using the restriction enzymes Nhe I and Xho I. The PCR product was then inserted into the vector with the help of T4 DNA ligase. A colony PCR was used to identify the recombinant plasmid from the transformed E. coli DH5α grown on an LB plate supplemented with kanamycin (50 mg/l). The plasmids from the PCR-positive colonies were further confirmed based on commercial DNA sequencing by BGI. The correct constructs were then chemically transformed into E. coli BL21(DE3) for IPTG-induced expression. All the operations were conducted according to classic molecular cloning methods [22] .
For expression, the conditions, including the cultivation temperature, IPTG concentration, and induction time, were all optimized. Briefly, the recombinant E. coli BL21 (DE3) was cultured in 5 ml of routinely autoclaved LB (with Kan) with shaking at 200 rpm and 37℃ overnight. One milliliter of the culture suspension was then transferred into 100 ml of LB with Kan. The culture conditions were kept unchanged. When the OD 600 of the culture reached 0.7, IPTG was added to a concentration of 0.5 mM to induce expression. The cultivation lasted for another 12 h at 20℃ with shaking. The induced cells were then harvested by centrifugation at 4℃ for 5 min at 10,000 × g and rinsed with a 20 mM Tris-HCl buffer (pH 7.4).
The purification process using a Ni-NTA matrix was conducted according to the manufacturer’s instructions. The cell pellet was resuspended in a binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4) and lysed by ultrasonication. Next, the lysate was separated by centrifugation (18,000 × g , 4℃, 10 min) and the supernatant then loaded onto an affinity column filled with Ni-NTA agarose 6FF and equilibrated with the binding buffer. The unbound proteins were removed by washing with a buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 7.4), and the target protein was washed with an elution buffer (20 mM Tris-HCl, 500 mM NaCl, 200 mM imidazole, pH 7.4). The eluent containing the target protein was then dialyzed against a dialysis buffer (50 mM Tris-HCl, pH 7.4) with magnetic stirring. The final concentration of the target protein was determined using the Bradford method.
The enzyme activity was determined based on the reduction of cofactor NAD + , the reduced form of which (NADH) was determined photospectromically at 340 nm [12] . The assay system was set as 6 mM ethanol, 0.2 mM NAD + , and an appropriate volume of the enzyme solution in a 50 mM Tris-HCl buffer (pH 8.8) [3] . The reaction mixture was pre-incubated for 10 min at 37℃ in a water bath. A reaction system without the enzyme was used as the blank. The activity was calculated based on the standard curve obtained with different solution concentrations of NADH. One unit (U) was defined as the amount of enzyme catalyzing the formation of 1 μmol of NADH per minute at 37℃ [14] .
- Activity with Various Hydroxyl-Group-Containing Substrates
A variety of hydroxyl-group-containing compounds ( L -threonine, D -threonine, L -serine, methanol, ethanol, n -butanol, glucose, isopropanol, glycerol, lactic acid, and citric acid) were tested as possible substrates for the enzyme. In these tests, NAD + was 0. 2mM and the concentration o f each substrate was 6 mM. The other conditions were the same as described above. The compound with which the enzyme exhibited the highest activity was used as the substrate in the subsequent experiments.
- Threonine Catabolism in Mutant MG1655 with Disrupted orf382
To confirm that the protein encoded by orf382 is indeed an L -threonine dehydrogenase, a mutant MG1655 with a disrupted orf382 was constructed using an RED recombination system by RUIDIBIO (Shanghai, China) [4] . It has already been reported that LTDH is the enzyme responsible for glycine synthesis from threonine in E. coli . Thus, to verify that the growth of the mutant was dependent on glycine, due to the disrupted orf382, the wild-type strain and mutant E. coli MG1655 were inoculated into two media, a minimal medium (glycerol, 20 g/l; disodium phosphate heptahydrate, 12.8 g/l; monopotassium phosphate, 3 g/l; ammonium chloride, 1 g/l; sodium chloride, 0.5 g/l; magnesium sulfate, 2 mM) with 1 mM glycine and a minimal medium without glycine. The cultures were incubated in 5 ml of the medium with shaking at 200 rpm and 37℃ overnight. The OD 600 was then measured as an indicator of growth.
- Effects of Temperature and pH on Activity, and Isoelectric Point
To determine the optimal temperature and pH of the enzyme, L -threonine (6 mM) was used as the substrate and NAD + (0.2 mM) as the cofactor. In the experiment to determine the optimum temperature, the assays were carried out using a temperature range from 16℃ to 45℃. After pre-incubation in a water bath, the A 340 of the reaction systems was determined. In general, the detectable activity of an enzyme is lowered at a high and low temperature. To ensure the tested values were in a reasonable range, the reaction time in this section was extended to 30 min.
To determine the optimal pH, a Tris-HCl buffer (50 mM) and glycine-NaOH buffer (50 mM) were used to provide an acidity range from pH 7.2 to 10.6. The enzymatic activity was assayed in the buffers with a specific pH, and the optimal pH was determined based on the buffer with the highest enzyme activity.
A spectrophotometric method was used to determine the isoelectric point (pI) of the enzyme. As the pI was predicted to be 5.2 using an online tool [5] ( ), only an acidic pH range was tested at a wavelength of 595 nm. Briefly, an equal volume of the enzyme solution was added to citric acid-Na 2 HPO 4 buffers (50 mM, pH 4~5.8.) and the absorbance was then recorded at 595 nm due to the formation of protein particles near the pI.
- Analysis of Metal Ions in Protein and Cofactor (NAD+, NADP+) Dependence
The purified and dialyzed protein sample was digested with HNO 3 . One milliliter (0.88 mg/ml) of the protein solution was then mixed with 4 ml of 16 M HNO 3 (guaranteed reagent) and shaken for 12 h at 25℃. Next, the sample was diluted to 10 ml with MilliQ water. The control without the protein was handled using the same procedure and same conditions [10] . The two samples were then assayed using inductively coupled plasma-atomic emission spectrometry (ICP-AES) [17] (types: Varian 710ES).
The cofactor dependence of the enzyme in the biocatalysis was tested using two reaction systems in the presence of 0.2 mM NAD + or NADP + . The enzymatic activity was then determined as previously described.
- Determination of Michaelis Constants
Based on the DNA sequence, the enzyme should have a molecular mass of 40 kDa. Therefore, in these tests, the concentration of the substrate was set from 1 to 10 μM for L -threonine and n -butanol, and 0.8 to 8 μM for ethanol. The concentration of NAD + was 10 μM. The temperature was maintained at 37℃ throughout the test; the absorbance at 340 nm was recorded at 10 min after the initiation of the reaction. The initial velocities were determined using a standard assay. All the data were fitted to a double-reciprocal plot based on which the Km values were then calculated [23] .
- Circular Dichroism Analysis
The secondary structure of the protein was analyzed based on the circular dichroism spectra (Chirascan, Applied Photophysics Ltd, UK) using the following settings: wavelength: 190-240 nm, step s ize: 1 nm, a nd t ime per point: 0. 5 sec. The s amples w ere prepared by diluting the protein solution to about 0.01 mg/ml in 20mM potassium phosphate (pH 7.4) [15] . The cell path was 10 mm. The data were processed using the online program [7] .
Results and Discussion
- Molecular Cloning, Protein Expression, and Purification
Using the sequence (Accession No. AAB18566.1) in GenBank as a reference, the orf382 was successfully amplified from the genomic DNA of E. coli MG1655, and inserted into the pET28a(+) plasmid between Nhe I and Xho I in a multiple cloning site ( Fig. 1 ). Sequencing confirmed the absence of any nucleotide mutation in the cloned sequence, implying that the expressed protein would share the same sequence with orf382 if it were to be efficiently expressed in the host E. coli BL(DE3). Based on the annotation in NCBI, the orf382 should encode an ADH (with a molecular mass of about 40 kDa) with a bound metal iron ion [25] . Therefore, in the subsequent experiments, this electronic inference was used as a guide for the expression and characterization studies.
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Structure of the recombinant plasmid carrying orf382 ready for expression. The recombinant plasmid was constructed by inserting the fragment orf382 cloned from the genome of E. coli MG1655 by PCR between the NheI and XhoI sites frequently used for protein fusion expression.
In the construct, induced expression should produce a protein with 6×His fused at its N-terminus. Here, the expression procedure was a routine operation in which an inducer was added to a concentration of 0.5 mM when the OD 600 reached 0.7. Based on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the molecular mass was consistent with the theoretical value of 40 kDa ( Fig. 2 ). However, disappointingly, about 50% of the target protein was insoluble. As the expression host was from the same strain lineage as the bacterium from which the protein was cloned, a codon bias would not have led to the insolubility of expressed target protein. One possible reason was that the increased expression level, even at a low temperature, was much higher than the background expression level in both strains. Owing to the observation of an insoluble part in the SDS-PAGE analysis, the induction was under a lower temperature of 20℃ when the target protein was purified for biochemical characterization.
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Expression and purification of the recombinant protein. As shown, the molecular mass of the LTDH was about 40 kDa. Lane 1: control for recombinant protein. Lane 2: whole cell expressing recombinant protein. Lane 3: supernate of cell lysate. Lane 4: precipitate of cell lysate. Lane 5: Protein MW Marker (Low) from TaKaRa. Lane 6: purified recombinant protein. Lane 7: protein in flow-through.
The target protein was purified from the lysate of the induced cells harboring the recombinant plasmid. First, its activity towards ethanol (6 mM) was tested in the presence of NAD + (0.2 mM). As a result, the overexpressed putative ADH was shown to catalyze the oxidation of ethanol based on an increased absorbance at 340 nm, corresponding to the generation of reduced NADH and indicating the enzymatic property of an alcohol dehydrogenase.
- Exploration of Substrate Spectrum
Although the enzyme was found to catalyze the oxidation of ethanol, a typical hydroxyl-group-containing compound ( i.e. , alcohol compound), the existence of different cellular components means it was also important to test the activity of the enzyme towards other similar compounds, especially those accessible to a bacterial cell or available in a cell [13] . Therefore, to explore a wider substrate spectrum, various hydroxyl-group-containing compounds, including L -threonine, D -threonine, L -serine, methanol, n -butanol, glucose, isopropanol, glycerol, lactic acid, and citric acid, were used as substrates for activity assays. The A 340 of the reaction system with a large amount of substrate was determined after incubating the enzyme solution for 10 min at a temperature of 37℃. As a result, since the recombinant protein exhibited a higher catalytic activity when using nbutanol, ethanol, and L -threonine as the substrates ( Fig. 3 ), it was considered reasonable to redesignate the protein as an L -threonine dehydrogenase, especially as normal E. coli cells have a very low chance of containing n -butanol. Moreover, previous reports have also indicated that it should be an LTDH enzyme.
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Substrate spectrum of the purified enzyme. 1. n-butanol; 2. L-threonine; 3. ethanol; 4. isopropanol; 5. glucose; 6. glycerol; 7. L-serine; 8. lactic acid; 9. citric acid; 10. methanol; 11. D-threonine.
- Growth of Mutant with Disrupted orf382 in Presence/Absence of Glycine
Based on the above results, it was expected that the mutant with a disrupted orf382 would not grow in a minimal medium without glycine supplementation, but would grow normally in a minimal medium with glycine supplementation; whereas the wild type would grow normally in both media. These predictions were verified by determining the population density of the two strains in the two growth environments. The growth of the mutant strain in the different environments experimentally confirmed the predicted growth phenotypes. Specifically, when glycine was absent, the mutant strain did not show any growth in the minimal medium ( Fig. 4 ), whereas the supplementation of heterogeneous glycine allowed the mutant strain to grow. Therefore, this study confirmed that the growth of the mutant with a disrupted orf382 was dependent on the addition of glycine, implying the function of orf382 in the catabolism of threonine into glycine. As such, the synthesis of glycine from threonine via threonine catabolism was seemingly blocked in the mutant strain owing to the disrupted orf382. Thus, the in vivo confirmation of the function of the protein encoded by orf382 provided experimental evidence for the proposed conclusion that the enzyme was an L -threonine dehydrogenase.
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Confirmation of the orf382 mutant. a. Wild type of E. coli MG1655. b. orf382 mutant of E. coli MG1655. The growth of the two strains was determined by measuring the OD600 after shaking at 200 rpm for 12 h at 37℃. The wild type grew normally in both media, whereas the mutant grew normally in the minimal medium with 1 mM glycine, but did not grow in the minimal medium without glycine supplementation.
- Optimal Temperature, pH, and Isoelectric Point
The temperature for the reaction systems was set in a range from 16℃ to 45℃ [14] , and the effect of the temperature on the enzyme activity is shown in Fig. 5 . In the experiments, the reaction speeded up when increasing the temperature, which continued up to a temperature of 39℃. Thereafter, the rate decreased rapidly, possibly due to thermal inactivation. Thus, the results indicated that the optimal temperature was 39℃, as shown in Fig. 5 . Meanwhile, the optimal pH of the enzyme was found to be pH 9.8 ( Fig. 6 ), which also matched with the results reported by Newman et al. [21] , where the optimal pH value was higher than 9.
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Determination of optimal temperature (Topt = 39℃). The assays were based on a set of temperature values ranging from 16℃ to 45℃ and were conducted as described in Materials and Methods, using L-Thr as the substrate. The reaction time was extended to 30 min to ensure the tested values were within a reasonable range.
The absorbance of a protein solution ( e.g. , A 595 ) near the isoelectric point of the protein is higher depending on the acidity of the buffer, owing to the formation of particles with a poorer solubility [29] . Therefore, the isoelectric point of the present enzyme was determined using a spectrophotometric method based on citric acid-Na 2 HPO 4 buffers (50 mM) with pH values ranging from 4 to 5.8 . As shown in Fig. 7 , the pI of the enzyme was about 5.4.
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Optimal pH (pH 9.8). Different buffers were used to determine the optimal pH in an oxidation reaction. The reactions were performed in a Tris-HCl buffer (50 mM) (●) and Gly-NaOH buffer (50 mM) (○), respectively, to provide an acidity range from pH 7.2 to 10.6, using 0.2 mM NAD+ and 6 mM L-threonine. The reactions were carried out at 37℃ for 10 min.
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Isoelectric point (pH = 5.4). The enzyme solution was mixed with an equal volume of citric acid-Na2HPO4 buffers (50 mM, pH 4~5.8.). The absorbance in these buffers was then recorded at 595 nm.
- Metal Ion Binding and Cofactor Dependence
The type and content of metal ions in the protein were assayed by ICP-AES. The same sample was used to test the contents of Fe and Zn ions, since Fe and Zn ions are frequently found in alcohol dehydrogenase. Surprisingly, the results showed 0.424 mol of Fe and 0.914 mol of Zn per mol of the protein. Since the coexistence of two kinds of ion in the same protein sample is unusual, one possible explanation is that the metal ion selectivity of the protein was not strict.
The enzyme activity in the two reaction systems was determined in the presence of cofactor NAD + or NADP + . The specific activity was 0.0145 U/mg and 0.0077 U/mg when using NAD + and NADP + , respectively. Thus, since NAD + supported twice the activity with NADP + , the enzyme exhibited an apparent preference for NAD + over NADP + [27] .
- Kinetic Studies
The initial velocities were determined in a standard assay at 37℃, and the data processing was performed using a Lineweaver-Burk plot [18] . The established linear relationship was then used to calculate the kinetic parameters of L -threonine, ethanol, and n -butanol. When NAD + was used as the cofactor, the K m for L -threonine, ethanol, and n -butanol was 11.29 μM (R 2 = 0.9977), 222.5 μM (R 2 = 0.9951), and 8.02 μM (R 2 = 0.9950), respectively. Therefore, when taking account of the presence of the tested substrates in the living E. coli cells, the results also suggested that the natural substrate could be L -threonine [9] .
- Circular Dichroism Analysis
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Circular dichroism analysis. The circular dichroism spectra for the samples were recorded on a Chirascan. Each spectrum was recorded within a range from 190 to 240 nm using a cuvette with a pathlength of 10 mm.
Finally, a circular dichroism analysis was used to investigate the secondary structure and folding properties of the recombinant protein [6] . Five different samples were set up and analyzed using circular dichroism. Meanwhile, the enzyme activity of the sample dialyzed against an EDTA solution was tested using the procedure described in the Materials and Methods [11] . The five samples were the protein without any treatment (S1); the protein incubated with the substrate 0.5 mM ethanol (S2), 0.5 mM L -threonine (S3), or 0.5 mM n -butanol (S4); and the protein dialyzed against EDTA (0.2 mg/ml protein was dialyzed in 20 mM potassium phosphate against a 5 × 10 -3 mM EDTA solution for 6 h and diluted to 0.01 mg/ml, S5). The circular dichroism spectra are shown in Fig. 8 . The specific activity of S5 was 0.001 U/mg, which was much lower (about 6.7%) than that of the enzyme without any treatment (0.015 U/mg). Therefore, this implies that the enzyme activity requires the presence of a metal ion. The circular dichroism plot also revealed a clear difference between S5 and the other samples. To quantify the changes in the secondary structure of the samples, the data were processed using an online program [7] ( ), as shown in Table 1 . The protein without any treatment exhibited a similar secondary structure distribution to the proteins incubated with the substrates. Owing to deprivation of metal ions with dialysis against an EDTA solution, the content of the α-helix was reduced from 12.6% to 6.3%; correspondingly, the secondary structure of a β-sheet, β-turn, and random coil increased by 0.5%, 2.1%, and 3.7%, respectively. Therefore, the results indicated that the substrate binding (samples S2, S3, and S4) did not markedly influence the secondary structure of the enzyme when compared with sample S1. However, when comparing the data for S1 and S5, the bound metal ion was clearly essential for the protein to maintain its proper secondary structure required for activity.
Data processing of circular dichroism spectra.
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S1: protein without any treatment. S2: protein with 0.5 mM ethanol. S3: protein with 0.5 mM L-threonine. S4: protein with 0.5 mM n-butanol. S5: protein dialyzed against EDTA.
This study was supported by a Key grant from the Chinese Education Ministry and partially supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, China.
Ben-Shitrit T , Yosef N , Shemesh K , Sharan R , Ruppin E , Kupiec M 2012 Systematic identification of gene annotation errors in the widely used yeast mutation collections. Nat. Methods 9 373 - 378    DOI : 10.1038/nmeth.1890
Cochrane G , Karsch-Mizrachi I , Nakamura Y 2011 The international nucleotide sequence database collaboration. Nucleic Acids Res. 39 D15 - D18    DOI : 10.1093/nar/gkq1150
Crow JP , Beckman JS , McCord JM 1995 Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite. Biochemistry 34 3544 - 3552    DOI : 10.1021/bi00011a008
Datsenko KA , Wanner BL 2000 One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97 6640 - 6645    DOI : 10.1073/pnas.120163297
Feng DD , Yang W , Yang WJ , Yu QY , Zhang Z 2012 Molecular cloning, heterogenous expression and the induction profiles after organophosphate phoxim exposure of the carboxylesterase Bmae33 in the silkworm, Bombyx mori. Afr. J. Biotechnol. 11 9915 - 9923
Greenfield NJ 2006 Analysis of the kinetics of folding of proteins and peptides using circular dichroism. Nat. Protoc. 1 2891 - 2899    DOI : 10.1038/nprot.2006.244
Greenfield NJ 2006 Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1 2876 - 2890    DOI : 10.1038/nprot.2006.202
Hayashi K , Morooka N , Yamamoto Y , Fujita K , Isono K , Choi S 2006 Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol. Syst. Biol. 2 2006 - 2007    DOI : 10.1038/msb4100049
Higashi N , Fukada H , Ishikawa K 2005 Kinetic study of thermostableL-threonine dehydrogenase from an archaeon Pyrococcus horikoshii. J. Biosci. Bioeng. 99 175 - 180    DOI : 10.1263/jbb.99.175
Högbom M , Lam R , Bakali HMA , Kuznetsova E , Nordlund P , Zamble DB 2005 A high throughput method for the detection of metalloproteins on a microgram scale. Mol. Cell Proteomics 4 827 - 834    DOI : 10.1074/mcp.T400023-MCP200
Jana S , Chaudhuri TK , Deb JK 2006 Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy. Biochem. (Mosc.) 71 1230 - 1237    DOI : 10.1134/S0006297906110083
Johnson AR , Dekker EE 1996 Woodward’s reagent K inactivation of Escherichia coliL-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups. Protein Sci. 5 382 - 390    DOI : 10.1002/pro.5560050223
Kaulmann U , Smithies K , Smith MEB , Hailes HC , Ward JM 2007 Substrate spectrum of ω-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis. Enzyme Microb. Technol. 41 628 - 637    DOI : 10.1016/j.enzmictec.2007.05.011
Kazuoka T , Takigawa S , Arakawa N , Hizukuri Y , Muraoka I , Oikawa T , Soda K 2003 Novel pychrophilic and thermolabileL-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1. J. Bacteriol. 185 4483 - 4489    DOI : 10.1128/JB.185.15.4483-4489.2003
Kelly SM , Jess TJ , Price NC 2005 How to study proteins by circular dichroism. Biochim. Biophys. Acta 1751 119 - 139    DOI : 10.1016/j.bbapap.2005.06.005
Knight R , Jansson J , Field D , Fierer N , Desai N , Fuhrman JA 2012 Unlocking the potential of metagenomics through replicated experimental design. Nat. Biotechnol. 30 513 - 520    DOI : 10.1038/nbt.2235
Li X , Coles BJ , Ramsey MH , Thornton I 1995 Sequential extraction of soils for multielement analysis by ICP-AES. Chem. Geol. 124 109 - 123    DOI : 10.1016/0009-2541(95)00029-L
Machielsen R , van der Oost J 2006 Production and characterization of a thermostableL-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. FEBS J. 273 2722 - 2729    DOI : 10.1111/j.1742-4658.2006.05290.x
Mardis ER 2011 A decade’s perspective on DNA sequencing technology. Nature 470 198 - 203    DOI : 10.1038/nature09796
Moon TS , Lou C , Tamsir A , Stanton BC , Voigt CA 2012 Genetic programs constructed from layered logic gates in single cells. Nature 491 249 - 253    DOI : 10.1038/nature11516
Newman EB , Kapoor V , Potter R 1976 Role ofL-threonine dehydrogenase in the catabolism of threonine and synthesis of glycine by Escherichia coli. J. Bacteriol. 126 1245 - 1249
Sambrook JR , Russell DW 2001 Molecular Cloning: A Laboratory Manual 3rd ed. Cold Spring Harbor Laboratory Press New York
Shimizu Y , Sakuraba H , Kawakami R , Goda S , Kawarabayasi Y , Ohshima T 2005 L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization. Extremophiles 9 317 - 324    DOI : 10.1007/s00792-005-0447-2
Sibbald MJ , Ziebandt AK , Engelmann S , Hecker M , de Jong A , Harmsen HJ 2006 Mapping the pathways to staphylococcal pathogenesis by comparative secretomics. Microbiol. Mol. Biol. Rev. 70 755 - 788    DOI : 10.1128/MMBR.00008-06
Sofia HJ , Burland V , Daniels DL , Plunkett G , Blattner FR 1994 Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes. Nucleic Acids Res. 22 2576 - 2586    DOI : 10.1093/nar/22.13.2576
Stower H 2013 Metagenomics: personalized gut microbiome variants. Nat. Rev. Genet. 14 80 -    DOI : 10.1038/nrg3409
Tressel T , Thompson R , Zieske LR , Menendez MI , Davis L 1986 Interaction betweenL-threonine dehydrogenase and aminoacetone synthetase and mechanism of aminoacetone production. J. Biol. Chem. 261 16428 - 16437
Turner JM 1967 Microbial metabolism of amino ketones.L-1-aminopropan-2-ol dehydrogenase andL-threonine dehydrogenase in Escherichia coli. Biochem. J. 104 112 - 121
Valnickova Z , Christensen T , Skottrup P , Thogersen IB , Hojrup P , Enghild JJ 2006 Post-translational modifications of human thrombin-activatable fibrinolysis inhibitor (TAFI): evidence for a large shift in the isoelectric point and reduced solubility upon activation. Biochemistry 45 1525 - 1535    DOI : 10.1021/bi051956v
Williamson SJ , Yooseph S 2012 From bacterial to microbial ecosystems (metagenomics). Methods Mol. Biol. 804 35 - 55
Xu J , Johnson RC 1995 aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. J. Bacteriol. 177 3166 - 3175