Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B.
Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A. Equilibrium unfolding analysis revealed that the DeltaG(U)(H(2)O) values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcal/mol, respectively, relative to that of the wild-type enzyme. The interface mutations not only decreased the k(cat)/K(M) value by about 8- to 96-fold, but also increased the K(D) value for d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold. The crystal structure of R72A determined at 2.5 A resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI. Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI.
Nam
Gyu Hyun
GH
National Research Laboratory of Protein Folding and Engineering, Pohang, South Korea.
Kim
Do-Hyung
DH
Ha
Nam-Chul
NC
Jang
Do Soo
DS
Yun
Young Sung
YS
Hong
Bee Hak
BH
Oh
Byung-Ha
BH
Choi
Kwan Yong
KY
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D019281:Dimerization; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013251:Steroid Isomerases / Q000737:chemistry* / Q000235:genetics*; D013816:Thermodynamics
D000596:Amino Acids; D011994:Recombinant Proteins; D013251:Steroid Isomerases