深海性Shewanella属細菌由来シトクロムcの安定化機構の解明

内容の要約広島大学(Hiroshima University)博士(農学)Doctor of Agriculturedoctora

Difference in NaCl tolerance of membrane-bound 5′-nucleotidases purified from deep-sea and brackish water Shewanella species

Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5′-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance in order to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl2 for their activities, and they exhibited the same pH dependency and substrate specificity for 5′-nucleotides. However, S. violacea 5′-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5′-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5′-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan [No. 26240045], a grant from the Japan Society for the Promotion of Science [No. 25-1446], and The Salt Science Research Foundation [No. 1655]

Structural and functional insights into thermally stable cytochrome c' from a thermophile

Thermophilic Hydrogenophilus thermoluteolus cytochrome c0 (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c0 (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 A ° . In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit–subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit–subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit–subunit interactions with conservation of the ligand-binding ability.This work was performed under the Cooperative Research Program of the “Network Joint Research Center for Materials and Devices”

Commonly stabilized cytochromes <i>c</i> from deep-sea <i>Shewanella</i> and <i>Pseudomonas</i>

<p>Two cytochromes <i>c</i>₅ (SBcyt<i>c</i> and SVcyt<i>c</i>) have been derived from <i>Shewanella</i> living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcyt<i>c</i> and SVcyt<i>c</i> were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcyt<i>c</i> and SVcyt<i>c</i> was higher than that of homologues from atmospheric-pressure <i>Shewanella</i>, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome <i>c</i>₅₅₁ (PMcyt<i>c</i>) of deep-sea <i>Pseudomonas</i> was more stable than a homologue of atmospheric-pressure <i>Pseudomonas aeruginosa</i>, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcyt<i>c</i> and SVcyt<i>c</i>, and PMcyt<i>c</i> are phylogenetically very distant, these deep-sea cytochromes <i>c</i> are commonly stabilized through hydrogen bond formation.</p> <p>Deep-sea cytochromes <i>c</i> are commonly stabilized through hydrogen bond formation.</p