Masaharu Ishii

Stabilization of Pseudomonas aeruginosa Cytochromec 551 by Systematic Amino Acid Substitutions Based on the Structure of Thermophilic Hydrogenobacter thermophilus Cytochrome c 552

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochromec 551 (PA c 551) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c 551 with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochromec 552 (HT c 552), which has similar main chain folding to PA c 551, but is more stable to heat. Thermodynamic properties of PAc 551 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 c 551 with HTc 552. 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 c 552. 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 PAc 551 can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c 552. The higher stability of HT c 552 may in part be attributed to some of these substitutions.

Phylum BI. Aquificae phy. nov.

Rod-shaped, moderately thermophilic to hyperthermophilic. Isolated from marine and terrestrial hydrothermal springs. Includes microaerophilic chemolithotrophic hydrogen oxidizers, and chemoorganotrophs. Most members have been reported to be motile. All members, with the exception of Hydrogenobacter acidophilum, grow best between pH 6.0 and 8.0.