Purification and Characterization of a Thermostable Class II Fumarase fromThermus thermophilus

doi:10.1006/abbi.1998.0693

Archives of Biochemistry and Biophysics

Volume 355, Issue 1, 1 July 1998, Pages 49-55

https://doi.org/10.1006/abbi.1998.0693 Get rights and content

Abstract

A thermostable fumarase was purified from a strain ofThermus thermophilusisolated from a Japanese hot spring. The maximum specific activity of the purified enzyme was 1740 units/mg at pH 8.0 and 85°C. The enzyme was composed of four identical subunits with a molecular weight of 46,000 and displayed other enzymatic characteristics which are common to the class II fumarases. The thermal stability of the purified enzyme was remarkable, with over 80% of the activity remaining after a 24-h incubation at 90°C. The enzyme was also resistant to chemical denaturants; 50% of the initial specific activity was detected in assay mixtures containing 0.8 M guanidine hydrochloride. The purified enzyme shared an extremely high sequence homology withThermus aquaticusfumarase andBacillus subtilisfumarase in the first 43 amino acid residues.

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Enhancing the thermostability of fumarase C from Corynebacterium glutamicum via molecular modification
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Citation Excerpt :
Some fumarases from thermophilic bacteria have good thermal stability and their optimum reaction temperature can be as high as 85 °C. However, When the reaction temperature of the enzymes from these thermophiles is reduced to 40–60 °C, the enzyme activity is only 10%–20% of the highest enzyme activity [12]. Moreover, it will consume enormous energy if maintain a reaction temperature of 85 °C when the production scale is large.
Fumarases have been successfully applied in industry for the production of l-malate. However, the industrialization of fumarases is limited by their low thermostability. In this study, the thermostability of fumarase C from Corynebacterium glutamicum was enhanced through directed evolution, simulated mutagenesis, site-directed mutagenesis and saturated mutagenesis. Mutant 2G (A411V) was initially constructed through directed evolution. Its half-life at 50 °C (t_1/2, _50°C) increased from 1 min to 2.2 min, and the T₅₀¹⁵ (temperature at which the activity of enzyme decreased by 50% in 15 min) increased from 44.8 °C to 47.2 °C. Besides, several different mutants were obtained by site-directed mutation. Among them, mutant 3G (A227V) showed significant improvement in thermostability with a 3.3-fold improvement of t_1/2, _50°C and a 3.6 °C increase in T₅₀¹⁵ compared to the wild-type enzyme. Then, 2/3G (A227V, A411V) was obtained by combining the mutant 2G with the mutant 3G, for which the t_1/2, _50°C and T₅₀¹⁵ increased to more than 768 min and 52.4 °C, respectively. Finally, site-saturated mutagenesis was employed on amino acid residues 175-Glu, 228-Gly, 297-Gly, 320-Lys and 464-Glu to maximize the thermostability of mutant 2/3G. The most thermostable mutant 175G with amino acid substitutions (A227V, A411V, E175K) was isolated. Its t_1/2,50°C increased to more than 2700 min while that of wild-type enzyme was only 1 min and T₅₀¹⁵ was 9.8 °C higher than the wild-type enzyme. The thermostable mutated enzymes generated without affecting the activity in this study would be an attractive candidate for industrial applications.
Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties
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Citation Excerpt :
The behavior of molar ellipticity as a function of temperature at 222 (Fig. 1B) and 220 nm (Fig. 1D) indicate melting temperatures of 56.3 °C for LmFH-1 and 57.7 °C for LmFH-2, respectively. The thermal stability observed implies that the LmFH isoforms can be considered thermolabile enzymes, especially when compared to the class 2 thermostable enzymes where the Tm values can reach 80–90 °C [29–31]. Comparing with the initial values, analysis of the spectral rescan after the complete run of thermal scan confirmed a significant loss of α-helix content and β-strand structure, and an increase in unordered content.
Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs.
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¹: To whom correspondence should be addressed at Department of Biotechnology, Faculty of Engineering, Tottori University, Koyama-Minami, Tottori 680-8552, Japan. Fax: +81-857-31-5271. E-mail:[email protected].

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Regular ArticlePurification and Characterization of a Thermostable Class II Fumarase fromThermus thermophilus

Abstract

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochim. Biophys. Acta

Anal. Biochem.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochemistry

J. Biochem.

Proc. Natl. Acad. Sci. USA

J. Gen. Microbiol.

J. Bacteriol.

Regular Article
Purification and Characterization of a Thermostable Class II Fumarase fromThermus thermophilus