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Disassembly of the ring-type decameric structure of peroxiredoxin from Aeropyrum pernix K1 by amino acid mutation.

ABSTRACT: The quaternary structure of peroxiredoxin from Aeropyrum pernix K1 (ApPrx) is a decamer, in which five homodimers are assembled in a pentagonal ring through hydrophobic interactions. In this study, we determined the amino acid (AA) residues of ApPrx crucial for forming the decamer using AA mutations. The ApPrx0Cys mutant, wherein all cysteine residues were mutated to serine, was prepared as a model protein to remove the influence of the redox states of the cysteines on its assembling behavior. The boundary between each homodimer of ApPrx0Cys contains characteristic aromatic AA residues forming hydrophobic interactions: F46, F80, W88, W210, and W211. We found that a single mutation of F46, F80, or W210 to alanine completely disassembled the ApPrx0Cys decamer to homodimers, which was clarified by gel-filtration chromatography and dynamic light scattering measurements. F46A, F80A, and W210A mutants lacked only one aromatic ring compared with ApPrx0Cys, indicating that the assembly is very sensitive to the surface structure of the protein. X-ray structures revealed two mechanisms of disassembly of the ApPrx decamer: loss of hydrophobicity between homodimers and flip of the arm domain. The AA residues targeted in this study are well conserved in ring-type Prx proteins, suggesting the importance of these residues in the assembly. This study demonstrates the sensitivity and modifiability of peroxiredoxin assembly by a simple AA mutation.

SUBMITTER: Himiyama T

PROVIDER: S-EPMC7184774 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Little is known regarding the DNA methyltransferases (MTases) in hyperthermophilic archaea. In this study, we focus on an MTase from Aeropyrum pernix K1, a hyperthermophilic archaeon that is found in hydrothermal vents and whose optimum growth temperature is 90°C to 95°C. From genomic sequence analysis, A. pernix K1 has been predicted to have a restriction-modification system (R-M system). The restriction endonuclease from A. pernix K1 (known as ApeKI from New England BioLabs Inc. [catalog code R06435]) has been described previously, but the properties of the MTase from A. pernix K1 (M.ApeKI) have not yet been clarified. Thus, we demonstrated the properties of M.ApeKI. In this study, M.ApeKI was expressed in Escherichia coli strain JM109 and affinity purified using its His tag. The recognition sequence of M.ApeKI was determined by methylation activity and bisulfite sequencing (BS-seq). High-performance liquid chromatography (HPLC) was used to detect the position of the methyl group in methylated cytosine. As a result, it was clarified that M.ApeKI adds the methyl group at the C-5 position of the second cytosine in 5'-GCWGC-3'. Moreover, we also determined that the MTase optimum temperature was over 70°C and that it is strongly tolerant to high temperatures. M.ApeKI is the first highly thermostable DNA (cytosine-5)-methyltransferase to be evaluated by experimental evidence. IMPORTANCE In general, thermophilic bacteria with optimum growth temperatures over or equal to 60°C have been predicted to include only N4-methylcytosine or N6-methyladenine as methylated bases in their DNA, because 5-methylcytosine is susceptible to deamination by heat. However, from this study, A. pernix K1, with an optimum growth temperature at 95°C, was demonstrated to produce a DNA (cytosine-5)-methyltransferase. Thus, A. pernix K1 presumably has 5-methylcytosine in its DNA and may produce an original repair system for the expected C-to-T mutations. M.ApeKI was demonstrated to be tolerant to high temperatures; thus, we expect that M.ApeKI may be valuable for the development of a novel analysis system or epigenetic editing tool.

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Disassembly of the ring-type decameric structure of peroxiredoxin from Aeropyrum pernix K1 by amino acid mutation.

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