Stabilization of Pseudomonas aeruginosa cytochrome c(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 holocytochrome c(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 cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(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 HT c(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 PA c(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.
Hasegawa
J
J
Daiichi Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo 134-8630, Japan. haseg7li@daiichipharm.co.jp
Shimahara
H
H
Mizutani
M
M
Uchiyama
S
S
Arai
H
H
Ishii
M
M
Kobayashi
Y
Y
Ferguson
S J
SJ
Sambongi
Y
Y
Igarashi
Y
Y
D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D019791:Guanidine; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001426:Bacterial Proteins; D003574:Cytochrome c Group; C083951:cytochrome c553; C017167:cytochrome C(551); C022302:cytochrome C-552; D019791:Guanidine