Fumarase From Cyanidioschyzon merolae Stably Shows High Catalytic Activity for Fumarate Hydration Under High Temperature Conditions

Abstract

Fumarases (Fums) catalyze the reversible reaction converting fumarate to l-malate. There are two kinds of Fums: Class І and ІІ. Thermostable Class ІІ Fums, from mesophilic microorganisms, are utilized for industrial l-malate production. However, the low thermostability of these Fums is a limitation in industrial l-malate production. Therefore, an alternative Class ІІ Fum that shows high activity and thermostability is required to overcome this drawback. Thermophilic microalgae and cyanobacteria can use carbon dioxide as a carbon source and are easy to cultivate. Among them, Cyanidioschyzon merolae and Thermosynechococcus elongatus are model organisms to study cell biology and structural biology, respectively. We biochemically analyzed Class ІІ Fums from C. merolae (CmFUM) and T. elongatus (TeFum). Both CmFUM and TeFum preferentially catalyzed fumarate hydration. The catalytic activity of CmFUM for fumarate hydration in the optimum conditions (52°C and pH 7.5) is higher compared to those of Class ІІ Fums from other organisms and TeFum. Thermostability tests of CmFUM revealed that CmFUM showed higher thermostability than those of Class ІІ Fums from other microorganisms. The yield of l-malate obtained from fumarate hydration catalyzed by CmFUM was 75-81%. In summary, CmFum has suitable properties for efficient l-malate production.

Keywords: cyanobacteria; fumarase; l-malate; microalgae; tricarboxylic acid cycle.

Figure 1

SDS-PAGE results after purification of GST-tagged CmFUM (left) and TeFum (right). SDS-PAGE gels (8%) were stained with InstantBlue (Expedion Protein Solutions, San Diego, CA).

Figure 2

Temperature and pH dependence of CmFUM and TeFum (A) CmFUM activity at each temperature. The measurements using fumarate and l-malate as substrates were performed at pH 7.5 and 8.5, respectively. The concentrations of fumarate and l-malate were 0.5 and 5 mM, respectively. (B) CmFUM activity at each pH level. The measurements were performed at 52°C. The concentrations of fumarate and l-malate were 0.5 and 5 mM, respectively. (C) TeFum activity at each temperature. The measurements using fumarate and l-malate as substrates were performed at pH 7.0 and 7.5, respectively. The concentrations of fumarate and l-malate were 0.5 and 1 mM, respectively. (D) TeFum activity at each pH level. The measurements were performed at 50°C. The concentrations of fumarate and l-malate were 0.5 and 1 mM, respectively. The circles and triangles in Figure 2 indicate the activity using fumarate and l-malate as substrates, respectively. All data in Figure 2 indicate the mean ± SD obtained from three independent experiments.

Figure 3

Saturation curves of CmFUM and TeFum for substrates. (A) Saturation curves of CmFUM for fumarate (circles) and l-malate (triangles). The measurements using fumarate and l-malate as substrates were performed at 52°C and pH 7.5, and 52°C and pH 8.5, respectively. (B) Saturation curves of TeFum for fumarate (circles) and l-malate (triangles). The measurements using fumarate and l-malate as substrates were performed at 50°C and pH 7.0, and 50°C and pH 7.5, respectively. All the data in Figure 3 indicate the mean ± SD obtained from three independent experiments.

Figure 4

Effects of three organic acids on CmFUM and TeFum activity. (A) CmFUM activity using fumarate (left) and l-malate (right) as a substrate in the presence of organic acids. The measurements using fumarate and l-malate as substrates were performed at 52°C and pH 7.5, and 52°C and pH 8.5, respectively. The concentrations of fumarate and l-malate were the K_m values of CmFUM, 0.27 mM and 1.49 mM, respectively. (B) TeFum activity using fumarate (left) and l-malate (right) as a substrate in the presence of organic acids. The measurements using fumarate and l-malate as substrates were performed at 50°C and pH 7.0, and 50°C and pH 7.5, respectively. The concentrations of fumarate and l-malate were the K_m values of TeFum, 0.14 mM and 0.20 mM, respectively. All organic acids used as effectors were sodium salts. All the enzymatic activities in Figure 4 are represented by relative activities and the activity in the absence of effectors (gray bar) was set at 100%. All data in Figure 4 indicate the mean ± SD obtained from three independent experiments. All asterisks in Figure 4 indicate statistically significant differences between the absence and presence of the effector (Welch’s t-test; ^*p < 0.05, ^**p < 0.01, ^***p < 0.005). All p-values obtained from Welch’s t-test in (A,B) are listed in Supplementary Tables S1 and S2, respectively.

Figure 5

Thermostability of CmFUM. (A) CmFUM activity after heat treatment at each temperature for 15 min. The measurements were performed at pH 7.5. The concentration of fumarate was 0.5 mM. The T₅₀ ¹⁵ was calculated using a linear equation obtained from six values (53–60°C). (B) CmFUM activity after heat treatment at 50°C for each time-point. The measurements were performed at pH 7.5. The concentration of fumarate was 0.5 mM. The t_1/2 was calculated using a linear equation obtained from all the values. All the enzymatic activities in Figure 5 are represented by residual activities, and the activity without heat-treatment was set at 100%. All the data in Figure 5 show the mean ± SD obtained from three independent experiments.

Figure 6

Effects of metal cations and buffer solutions on CmFUM activity. (A) CmFUM activity in the presence of 5 mM metal cations. The measurement was performed at 52°C and pH 7.5. The concentration of fumarate was the K_m of CmFUM, 0.27 mM. Ca: CaCl₂, Mg: MgCl₂, Na: NaCl, K: KCl (B) CmFUM activity in 100 mM buffer solutions. The measurement was performed at 52°C and pH 7.5. The concentration of fumarate was 0.5 mM. The asterisk indicates a statistically significant difference between the activity in Tris-HCl and HEPES-NaOH buffer (Welch’s t-test; ^***p < 0.005). All p-values obtained from Welch’s t-test in (A,B) are listed in Supplementary Tables S3 and S4, respectively.

Figure 7

The yield of l-malate obtained from fumarate hydration catalyzed by CmFUM. (A) The yield of l-malate when using 200 mM fumarate as a substrate. The concentration of CmFum was 1 μM. The measurement was performed at 52°C and pH 7.5. (B) The yield of l-malate when using 1 M fumarate as a substrate. The concentration of CmFum was 0.5 μM. The measurement was performed at 52°C and pH 7.5 for 24 h. All the data in Figure 7 show the mean ± SD obtained from three independent experiments.

Figure 8

Phylogenetic analysis of biochemically characterized Class ІІ Fums. Sequence alignment of 14 Class ІІ Fums was performed using CLC Sequence Viewer ver. 8.0. A phylogenetic tree was generated by maximum likelihood method based on 423 conserved amino acid residues using PhyML online (http://www.atgc-montpellier.fr/phyml/). Bootstrap value obtained by 500 replications indicates the reliability of each branch.

Figure 9

Amino acid sequence comparison of biochemically characterized Class ІІ Fums. Sequences of 14 Class ІІ Fums were obtained from GenBank and aligned using CLC Sequence Viewer ver. 8.0. The blue squares represent amino acid residues equivalent to position 257 and 441 of CmFUM which contribute to the activity of Class ІІ Fum from S. coelicolor (Lin et al., 2007). The green square represents an amino acid residue equivalent to position 401 of CmFUM which contributes to the activity of Class ІІ Fum from Synechocystis 6803 (Katayama et al., 2019). The red squares represent amino acid residues equivalent to position 268, 320, and 503 of CmFUM which contribute to the thermostability of Class ІІ Fum from C. glutamicum (Lin et al., 2018). The yellow square represents a loop region containing the sequence GSSxxPxKxN (called a SS loop) which contributes to substrate binding and catalytic activity (Puthan Veetil et al., 2012). AtFUM: Class ІІ Fum from A. thaliana, HsFUM: Class ІІ Fum from H. sapience, RoFUM: Class ІІ Fum from R. oryzae, ScFUM: Class ІІ Fum from S. cerevisiae, SyFum: Class ІІ Fum from Synechocystis 6803, EcFum: Class ІІ Fum from E. coli, TtFum: Class ІІ Fum from T. thermophilus, SsFum: Class ІІ Fum from S. solfataricus, SttFum: Class ІІ Fum from S. thermovulgaris, StlFum: Class ІІ Fum from S. lividans, StcFum: Class ІІ Fum from S. coelicolor, CgFum: Class ІІ Fum from C. glutamicum.