Gene cloning, expression, and enzyme purification
To prepare recombinant enzymes encoded by Tne genes CTN_0257, CNT_0580, CTN_1548, CTN_1655 and CTN_1756, these genes were amplified from Tne genomic DNA, and successfully cloned into E. coli expression vector pHsh to generate plasmids pHsh-0257, pHsh-0580, pHsh-1548, pHsh-1655, and pHsh-1756. The target genes in the expression plasmids were sequenced and it was confirmed that no mutation had occurred in them. All the expression plasmids were transformed into E. coli and the target genes were successfully induced to express in the recombinant cells (Fig. 1).
The recombinant proteins produced from Tne genes CTN_0257, CNT_0580, CTN_1548, CTN_1655, and CTN_1756 were designated as Zn-Adh, Fe-AAdh, Aldh, Fe-Adh1, and Fe-Adh2, based on their activities and metal ions, and the gene products had subunits similar to their putative molecular masses of 43.3, 42.6, 51.8, 40.6 and 43.6 kD, respectively (Fig. 1). These enzymes were purified to near gel electrophoresis homogeneity after heat treatment and metal affinity chromatography using Ni-column at room temperature (Fig. 1). However, the purified Fe-AAdh, Fe-Adh1, and Fe-Adh2 became inactivated within about a week after they were stored at -20 °C, implying that they were sensitive to O2 in the air and buffer. This problem was resolved by purifying these enzymes in an anaerobic chamber using degassed buffers and keeping the purified enzyme in sealed anaerobic serum tubes.
Intracellular conditions in the growing Tne cells
Biochemical characterization of these enzymes can reveal properties and potentials of each enzyme. However, the conditions in a living cell are equilibrated for a sustainable life, and most intracellular conditions do not meet the optimal reaction conditions of an enzyme. It is important to estimate how the enzymes catalyze energy metabolisms under physiological conditions in cells growing at about 80 °C and pH 7.0 or lower, where the reduced and oxidized forms of cofactors are present at the same time to support both the forward and reverse reactions in the cytoplasm.
The pH value and cofactor concentrations of NAD+, NADH, NADP+, and NADPH are usually limited to certain levels. Under chemostatic conditions, NAD+, NADH, NADP+, and NADPH concentrations determined for Clostridium acetobutylicum cells were 6.8, 0.97, 0.41, and < 0.2 µmol/g of dry cells, which are 1.30, 0.19, 0.08, and 0.04 µmol/g of wet cells, respectively [12, 15]. In this work, the pH value of nonbuffered Tne cell extracts was 6.5, which is might be approximately the intracellular pH of the cells in middle exponential phase of growth. The intracellular concentrations of NAD+, NADH, NADP+, and NADPH were determined as 1.8, 0.28, 0.18, and 0.06 mM in Tne cells in late logarithmic phase (Fig. 2a).
The concentration of substrates is an important parameter affecting enzyme activity. However, currently no method is known for determining the intracellular concentrations of the substrates, such as ac-CoA and ac-ald, for the final steps of ethanol fermentation pathway. Instead, we determined the tolerance of Tne cells to ac-ald and ethanol, which might indicate how much of those substances would inhibit cell growth. The tests revealed that the growth rate of Tne cells was reduced by 50% in the presence of about 1.7 mM ac-ald, while 120 mM ethanol in the medium reduced growth rate by about 25% (Fig. 2b). These results indicate that Tne can tolerate certain amount of ethanol, but it is very sensitive to ac-ald, which must be transiently reduced or oxidized in living cells.
Biochemical properties of the recombinant enzymes
Four possible reactions can occur in the final steps of ethanol formation from acetyl-CoA. To determine the number of steps each dehydrogenase can catalyze, the biochemical properties of the enzymes were characterized at their optimal reaction pH and temperatures, where substrate(s) and cofactor concentrations were approximately 10 times higher than the Km value of the enzyme. Among the five recombinant enzymes, Tne Aldh encoded by gene CTN_1548 catalyzed the oxidation of various aldehydes using either NAD+ or NADP+ as coenzyme, while it did not show any ac-CoA reduction activity. The main biochemical properties of recombinant enzymes encoded by Tne genes CTN_0257, CNT_0580, CTN_1655 and CTN_1756 are listed in Table 1.
Table 1
Optimal reaction conditions and cofactor dependences for the enzymes of hyperthermophilic ethanol fermentation pathway.
Enzyme (gene) | Fe-AAdh (CNT_0580) | Fe-Adh1 (CTN_1655) | Fe-Adh2 (CTN_1756) | Zn-Adh (CTN_0257) |
Reaction | Ac-CoA→Ac-ald | Ac-CoA←Ac-ald | Ac-ald→Eth | Ac-ald←Eth | Ac-ald→Eth | Ac-ald←Eth | Ac-ald→Eth | Ac-ald←Eth | Ac-ald→Eth | Ald←Eth |
pHopt | 7.0 | 9.0 | 7.0 | 8.5 | 8.5 | 8.5 | 7.0 | 8.5 | - | 5.8 |
Topt | 85℃ | 85℃ | 80℃ | 95℃ | 95℃ | 95℃ | 85℃ | 100℃ | - | 95℃ |
Spec activity | | | | | | | | | | |
NADPH | 0.92 ± 0.03 | | 3.27 ± 0.05 | | 0.70 ± 0.05 | | 3.09 ± 0.06 | | ND | |
NADH | ND | | 0.52 ± 0.06 | | ND | | ND | | ND | |
NADP | | 0.41 ± 0.01 | | 2.44 ± 0.04 | | 0.43 ± 0.05 | | 1.20 ± 0.06 | | 0.46 ± 0.03 |
NAD | | 0.16 ± 0.02 | | 0.36 ± 0.05 | | 0.22 ± 0.04 | | 0.25 ± 0.04 | | 0.30 ± 0.02 |
Ac-CoA, acetyle-CoA; Ac-ald, acetaldehyde; Eth, ethanol; ND, activity not detectable. |
Table 1 Optimal reaction conditions and cofactor dependences for the enzymes of hyperthermophilic ethanol fermentation pathway. (Attached file)
When searching for an enzyme that could reduce ac-CoA to ac-ald, we found that purified enzyme encoded by CNT_0580 was a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that could catalyze all the forward and reverse reactions in the pathway from ac-CoA to ethanol under optimal reaction conditions (Table 1). Fe-AAdh catalyzed forward reactions from Ac-CoA to ethanol optimally at pH 7.0 and the reverse reactions at pH 8.5 or higher (Fig. 3a). Fe-Adh1 and Fe-Adh2 catalyzed the forward and reverse reactions between ac-ald and ethanol without showing any ac-CoA reduction activity. However, the activity of Fe-Adh1 was much lower than that of Fe-Adh2. In comparison, Fe-Adh1 not only had lower activities, but also had higher pH and temperature optima than Fe-AAdh and Fe-Adh2 (Table 2). Therefore, Fe-Adh2 and Fe-AAdh were the main reactive enzymes for ethanol formation at the pH range that was approximately the intracellular pH of the middle exponential phase of cell growth (Fig. 3b). The Tne Zn-Adh specifically catalyzed the reaction from ethanol to ac-ald, and no activity was detected for the reaction from ac-ald to ethanol. This result is different from almost all the other Adhs reported previously [11, 12, 16]. The activities of these enzymes depended highly on the pH conditions of the reaction mixtures. Tne Zn-Adh reacted optimally at pH 5.8, which was close to pH 6.5, the pH value of non-buffered cell-free extracts of Tne. The CoA-independent Aldh had an optimal pH of 9.5, which together with Zn-Adh could form a detoxification system for the oxidation of ethanol and aldehyde.
Table 2
Kinetics of the enzymes under imitative physiological conditions at 80 ℃ and pH 6.5.
Enzyme (gene) | Fe-AAdh (CTN_0580) | Fe-Adh1 (CTN_1655) | Fe-Adh2 (CTN_1756) | Zn-Adh (CTN_0257) |
Reaction | Reduction | Oxidation | Reduction | Oxidation | Reduction | Oxidation | Reduction | Oxidation | Reduction | Oxidation |
Reaction | Ac-CoA→Ac-ald | Ac-CoA←Ac-ald | Ac-ald→Eth | Ac-ald←Eth | Ac-ald→Eth | Ac-ald←Eth | Ac-ald→Eth | Ac-ald←Eth | Ac-ald→Eth | Ac-ald←Eth |
Co-factor(s) a | NADPH b | Mixture | Mixture | Mixture | Mixture | Mixture | Mixture | Mixture | Mixture | |
Substrate | Ac-CoA | Ac-ald + CoA | Ac-ald b | Eth | Ac-ald | Eth | Ac-ald b | Eth | Ac-ald | Eth b |
Km (mM) | 0.23 ± 0.02 | ND | 1.24 ± 0.03 | ND | ND | ND | 2.75 ± 0.06 | ND | ND | 80.7 ± 1.68 |
Vmax (U/mg) | 0.26 ± 0.01 | ND | 2.49 ± 0.22 | ND | ND | ND | 2.79 ± 0.04 | ND | ND | 0.58 ± 0.01 |
Ac-CoA, acetyle-CoA; Ac-ald, acetaldehyde; Eth, ethanol; ND, activity not detectable. |
a Cofactor mixture, 1.8 mM NAD, 0.28 mM NADH, 0.18 mM NADP, and 0.06 mM NADPH. |
b These substrates were used in various concentrations for determining Km and Vmax values. |
Properties of Fe-AAdh in catalyzing ac-CoA reduction
Fe-AAdh was the first enzyme found in hyperthermophiles that catalyzed the reduction of ac-CoA to produce ac-ald. In addition to its reaction optima (Table 1), this enzyme was subjected to further characterization. Fe-AAdh had 1-h half-life at about 92 °C, which is a typical property of hyperthermophilic enzymes (Fig. 3c). When analyzed at 85 °C and pH 6.5, the activity of Fe-AAdh to catalyze ac-CoA reduction strongly depended on the concentration of NADPH. Although its specific activity was upto 0.92 at pH 7.0 (Table 1), the apparent Km and Vmax were 0.23 mM NADPH and 0.26 U/mg at pH 6.5 in the absence of NADH and NAD(P)+, and there was significant substrate inhibition when NADPH concentration exceeded 0.4 mM (Fig. 3d). At the imitated physiological concentrations of cytoplasm for the mixture of NAD(P)H and NAD(P)+, Fe-AAdh did not show activity in catalyzing ac-CoA reduction. It could be because 0.06 mM NADPH was a low concentration in the cells, which was detected after the reducing power had been dumped via H2 formation. Furthermore, the presence of NADH, NAD+, or NADP+ affected ac-CoA reduction activity by Fe-AAdh; the activity was increased by NADH and inhibited by NAD+ and NADP+ at physiological concentrations (Fig. 3e). The presence of the complex of 0.28 mM NADH, 1.8 mM NAD+, and 0.18 mM NADP+ resulted in a net decrease of Vmax (0.17 U/mg) along with an increase of Km (0.55 mM) (Fig. 3d).
Conversion of ac-ald to ethanol under imitative physiological conditions
To estimate the physiological roles of Fe-AAdh, Fe-Adh1, Fe-Adh2, and Zn-Adh in growing cells, enzyme activities were evaluated under physiological conditions similar to those in cytoplasm, which contained mixed cofactors (1.8 mM NAD+, 0.28 mM NADH, 0.18 mM NADP+, and 0.06 mM NADPH), pH 6.5, with 2 mM ac-CoA, ac-ald, or CoASH. In biochemical characterization under optimized conditions, Fe-AAdh, Fe-Adh1 and Fe-Adh2 exhibited significant activities in both ac-ald reduction (forward) and ethanol oxidation (reverse) reactions. However, under the imitative physiological conditions, all these three enzymes did not have significant activity to catalyze the reverse reaction between ac-ald and ethanol (Table 2). Therefore, Zn-Adh was the enzyme only able to catalyze the reduction of ethanol to ac-ald with a Km of 80 mM ethanol under imitative physiological conditions (Table 3), indicating that the physiological role of this enzyme is likely the catalysis of detoxification reaction when ethanol concentration is high.
Table 2 Kinetics of the enzymes under imitative physiological conditions at 80 ℃, pH 6.5. (Attached file)
For the forward reaction of ac-ald to ethanol, Fe-Adh1 did not have any detectable activity under imitative physiological conditions, probably because its activity was weak and its optimal reaction pH is much higher than the pH value detected in non-buffered cell-free extract. Therefore, Fe-AAdh and Fe-Adh2 are the main enzymes catalyzing the reduction of ac-ald. They might contribute equally to ethanol formation because they showed similar Vmax. However, Fe-AAdh showed a lower Km values for its substrate ac-ald compared to that of Fe-Adh2 (Table 2). Thus Fig. 3f summarizes the main roles of these enzymes in ethanol metabolic pathway as revealed through the present study.
In vitro ethanol formation
To confirm their physiological roles, Fe-AAdh, Fe-Adh1, Fe-Adh2, and Zn-Adh were used for in vitro formation of ethanol from ac-CoA. In the tests using ac-CoA and NADPH as substrates, ethanol was detected at new peaks in HPLC and GC profiles after Fe-AAdh catalysis (Fig. 4). The effects of alcohol dehydrogenases on ethanol formation were determined by adding Fe-Adh1, Fe-Adh2, or Zn-Adh into the reaction mixture of Fe-AAdh. Interestingly, all the three alcohol dehydrogenases, including Zn-Adh that showed activity only for ethanol oxidation, enhanced ethanol formation from ac-CoA (Fig. 4). The addition of 19 µg Fe-Adh2 into the reaction mixture of 47 µg Fe-AAdh resulted in 9.5 times increase of ethanol formation from ac-CoA, where ac-CoA reduction was catalyzed by Fe-AAdh alone and ac-ald reduction could be catalyzed by both Fe-AAdh and Fe-Adh2 (Table 3). These results reveal the complicate interactions of these enzymes in a biological system, and there is a strong synergy between the aldehyde and alcohol dehydrogenase activities. To explain why Zn-Adh, an enzyme did not show any acetaldehyde reduction activity, could have any stimulation of the ethanol formation, and how Fe-Adh2 stimulate ethanol production by 9.5 times, new methods are required for a further research.
Table 3
In vitro catalysis of ethanol formation from acetyl-CoA
Enzyme(s) | Fe-AAdh | Fe-AAdh + Fe-Adh1 | Fe-AAdh + Fe-Adh2 | Fe-AAdh + Zn-Adh |
Peak area | 1074 ± 126 | 1147 ± 180 | 5507 ± 236 | 4604 ± 356 |
Ethanol titer (mM) | 0.081 ± 0.020 | 0.092 ± 0.028 | 0.773 ± 0.037 | 0.633 ± 0.056 |
Relative activity (%) | 100 | 113 | 954 | 781 |
Reaction mixture (100 µl) contains 5 mM acetyl-CoA, 5 mM NADPH, 100 mM MOPS at pH 7.5, and 47 µg Fe-AAdh with or without addition of Fe-Adh1 (22 µg), Fe-Adh2 (19 µg), or Zn-Adh (43 µg). |
Bioinformatics of Fe-AAdh protein
The amino acid sequence of Fe-AAdh was subjected to the multiple sequence alignment (MSA) using the program T-Coffee and ESPript (Easy Sequencing in PostScript, http://espript.ibcp.fr/ESPript/ESPript/index.php) [17, 18]. The results revealed that the same enzyme appeared in hyperthermophilic Thermotoga spp. and archaea species (Table 4), which is annotated as iron-containing alcohol dehydrogenase (ADH). The amino acid sequences were also compared between Fe-AAdh and the other bifunctional aldehyde/alcohol dehydrogenases, AdhB an AdhE (Fig. 5). Interestingly, AdhE is a large protein with two domains of Fe-ADH (PF00465 family) and an ALDH (ALDH-SF superfamily) while bifunctional enzymes of Fe-AAdh and AdhB are similar to a monofunctional ADH such as Fe-Adh2 (Fig. 5). Meanwhile, Fe-AAdh and AdhB belong to the iron- and zinc-containing alcohol dehydrogenase families, respectively. These results indicate that these enzymes employ different strategies to achieve CoA-dependent aldehyde dehydrogenase activities.
Table 4
Distribution of Fe-AAdh in hyperthermophiles
Microorganism | GenBank accessory No. | Pfam family | Sequence similarity |
Thermotoga neapolitana DSM 4359 | CTN_0580 | PF00465 (Fe-ADH) | 100% |
Thermotoga maritima MSB8 | TM_0111 | PF00465 (Fe-ADH) | 100% |
Thermotoga sp. RQ7 | TRQ7_04415 | PF00465 (Fe-ADH) | 100% |
Thermotoga petrophila | Tpet_0813 | PF00465 (Fe-ADH) | 99% |
Pyrococcus furiosus DSM 3638 | PF_0075 | PF00465 (Fe-ADH) | 94% |
Thermococcus kodakarensis (ATCC BAA-918) | TK_RS07830 | PF00465 (Fe-ADH) | 93% |