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Crystal structure of thermally stable homodimeric cytochrome c'-β from Thermus thermophilus.

ABSTRACT: Cytochrome c'-β is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel β-sheet fold. Here, the crystal structure of cytochrome c'-β from the thermophilic Thermus thermophilus (TTCP-β) is reported at 1.74 Å resolution. TTCP-β has a typical antiparallel β-sheet fold similar to that of cytochrome c'-β from the moderately thermophilic Methylococcus capsulatus (MCCP-β). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-β and MCCP-β, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-β exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-β. Mutational analysis reveals that the increased homodimeric interface area of TTCP-β contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-β loop regions, possibly contribute to the rigid loop structure compared with MCCP-β, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-β, including TTCP-β, are optimized for function under the high-temperature conditions in which the source organisms live.

SUBMITTER: Yoshimi T

PROVIDER: S-EPMC9158659 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Crystal structure of thermally stable homodimeric cytochrome c'-β from Thermus thermophilus.

Yoshimi Taisuke T Fujii Sotaro S Oki Hiroya H Igawa Takeshi T Adams Hannah R HR Ueda Kengo K Kawahara Kazuki K Ohkubo Tadayasu T Hough Michael A MA Sambongi Yoshihiro Y

Acta crystallographica. Section F, Structural biology communications 20220527 Pt 6

Cytochrome c'-β is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel β-sheet fold. Here, the crystal structure of cytochrome c'-β from the thermophilic Thermus thermophilus (TTCP-β) is reported at 1.74 Å resolution. TTCP-β has a typical antiparallel β-sheet fold similar to that of cytochrome c'-β from the moderately thermophilic Methylococcus capsulatus (MCCP-β). The phenylalanine cap structure around the distal side ...[more]

PMID: 35647678

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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 thermally stable homodimeric cytochrome c'-β from Thermus thermophilus.

Publications

Crystal structure of thermally stable homodimeric cytochrome c'-β from Thermus thermophilus.

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