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Crystal structure of Thermus thermophilus methylenetetrahydrofolate dehydrogenase and determinants of thermostability.

ABSTRACT: The elucidation of mechanisms behind the thermostability of proteins is extremely important both from the theoretical and applied perspective. Here we report the crystal structure of methylenetetrahydrofolate dehydrogenase (MTHFD) from Thermus thermophilus HB8, a thermophilic model organism. Molecular dynamics trajectory analysis of this protein at different temperatures (303 K, 333 K and 363 K) was compared with homologous proteins from the less temperature resistant organism Thermoplasma acidophilum and the mesophilic organism Acinetobacter baumannii using several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis. These methods enabled the determination of important residues for the thermostability of this enzyme. The description of rotamer distributions by Gini coefficients and Kullback-Leibler (KL) divergence both revealed significant correlations with temperature. The emerging view seems to indicate that a static salt bridge/charged residue network plays a fundamental role in the temperature resistance of Thermus thermophilus MTHFD by enhancing both electrostatic interactions and entropic energy dispersion. Furthermore, this analysis uncovered a relationship between residue mutations and evolutionary pressure acting on thermophilic organisms and thus could be of use for the design of future thermostable enzymes.

SUBMITTER: Maiello F

PROVIDER: S-EPMC7219735 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Crystal structure of Thermus thermophilus methylenetetrahydrofolate dehydrogenase and determinants of thermostability.

Maiello Fernando F Gallo Gloria G Coelho Camila C Sucharski Fernanda F Hardy Leon L Würtele Martin M

PloS one 20200513 5

The elucidation of mechanisms behind the thermostability of proteins is extremely important both from the theoretical and applied perspective. Here we report the crystal structure of methylenetetrahydrofolate dehydrogenase (MTHFD) from Thermus thermophilus HB8, a thermophilic model organism. Molecular dynamics trajectory analysis of this protein at different temperatures (303 K, 333 K and 363 K) was compared with homologous proteins from the less temperature resistant organism Thermoplasma acido ...[more]

PMID: 32401802

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Project description:BackgroundMethylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.Methodology/principal findingsMTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the β(8)α(8) barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions.Conclusions/significanceThe different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.

Dataset Information

Crystal structure of Thermus thermophilus methylenetetrahydrofolate dehydrogenase and determinants of thermostability.

Publications

Crystal structure of Thermus thermophilus methylenetetrahydrofolate dehydrogenase and determinants of thermostability.

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