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]

Selected Mutations in a Mesophilic Cytochrome c Confer the Stability of a Thermophilic Counterpart

Mesophilic cytochrome c551 of Pseudomonas aeruginosa (PA c551) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c552 (HT c552), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c552 through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c551 could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c552, there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.This work was supported by a grant from the Japanese Ministry of Education, Science and Culture

Stabilization of Pseudomonas aeruginosa Cytochrome c551 by Systematic Amino Acid Substitutions Based on the Structure of Thermophilic Hydrogenobacter thermophilus Cytochrome c552

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c551 (PA c551) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c551 with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c552 (HT c552), which has similar main chain folding to PA c551, but is more stable to heat. Thermodynamic properties of PA c551 with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c551 with HT c552. In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c552. We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c551 can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c552. The higher stability of HT c552 may in part be attributed to some of these substitutions.This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture

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”

Mutants of Escherichia coli lacking disulphide oxdoreductases DsbA and DsbB cannot synthesise an exogenous monohaem c-type cytochrome except in the presence of disulphide compounds

Absence through mutation of two proteins involved in periplasmic disulphide bond formation, DsbA and DsbB, results in failure of anaerobically grown Escherichia coli to synthesise the holo forms of either its endogenous c-type cytochrome nitrite reductase or exogenous cytochrome c550 from Paracoccus denitrificans. The synthesis of both cytochromes can be restored to the mutants by inclusion in the growth media of compounds containing disulphide bonds, e.g., the oxidised form of glutathione. The results suggest that the attachment of haem to the CXXCH motif of a periplasmic c-type cytochrome may be preceded by the formation of one or more intra- or intermolecular disulphide bonds involving the cysteine residues of this motif.</p

Biological nano motor, ATP synthase FoF1: from catalysis to γϵc10–12 subunit assembly rotation

AbstractProton translocating ATPase (ATP synthase), a chemiosmotic enzyme, synthesizes ATP from ADP and phosphate coupling with the electrochemical ion gradient across the membrane. This enzyme has been studied extensively by combined genetic, biochemical and biophysical approaches. Such studies revealed a unique mechanism which transforms an electrochemical ion gradient into chemical energy through the rotation of a subunit assembly. Thus, this enzyme can be defined as a nano motor capable of coupling a chemical reaction and ion translocation, or more simply, as a protein complex carrying out rotational catalysis. In this article, we briefly discuss our recent work, emphasizing the rotation of subunit assembly (γϵc10–12) which is formed from peripheral and intrinsic membrane subunits

Effects of heme on the thermal stability of mesophilic and thermophilic cytochromes c: Comparison between experimental and theoretical results.

We have recently proposed a measure of the thermal stability of a protein: the water-entropy gain at 25 °C upon folding normalized by the number of residues, which is calculated using a hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach. A protein with a larger value of the measure is thermally more stable. Here we extend the study to analyses on the effects of heme on the thermal stability of four cytochromes c (PA c(551), PH c(552), HT c(552), and AA c(555)) whose denaturation temperatures are considerably different from one another despite that they share significantly high sequence homology and similar three-dimensional folds. The major conclusions are as follows. For all the four cytochromes c, the thermal stability is largely enhanced by the heme binding in terms of the water entropy. For the holo states, the measure is the largest for AA c(555). However, AA c(555) has the lowest packing efficiency of heme and the apo polypeptide with hololike structure, which is unfavorable for the water entropy. The highest stability of AA c(555) is ascribed primarily to the highest efficiency of side-chain packing of the apo polypeptide itself. We argue for all the four cytochromes c that due to covalent heme linkages, the number of accessible conformations of the denatured state is decreased by the steric hindrance of heme, and the conformational-entropy loss upon folding becomes smaller, leading to an enhancement of the thermal stability. As for the apo state modeled as the native structure whose heme is removed, AA c(555) has a much larger value of the measure than the other three. Overall, the theoretical results are quite consistent with the experimental observations (e.g., at 25 °C the α-helix content of the apo state of AA c(555) is almost equal to that of the holo state while almost all helices are collapsed in the apo states of PA c(551), PH c(552), and HT c(552))