11 results on '"Hyung Jin Cha"'
Search Results
2. Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase
- Author
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Jae Sung Woo, Do Soo Jang, Yeon Gil Kim, and Kyong-Tai Kim
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Protein Folding ,Mutant ,Steroid Isomerases ,Isomerase ,Crystallography, X-Ray ,medicine.disease_cause ,Hydrophobic effect ,chemistry.chemical_compound ,Isomerism ,Catalytic Domain ,Ketosteroid ,Enzyme Stability ,medicine ,Urea ,Molecular Biology ,Mutation ,biology ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,Articles ,Cell Biology ,General Medicine ,Kinetics ,Amino Acid Substitution ,chemistry ,Biochemistry ,Biocatalysis ,Biophysics ,biology.protein ,Mutant Proteins ,Protein folding ,Function (biology) - Abstract
Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 Å resolution, respectively, and compared with previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a second-site suppressor.
- Published
- 2013
3. Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase
- Author
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Jae-Hee Jeong, Eun Ju Shin, Young Sung Yun, and Do Soo Jang
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0301 basic medicine ,Stereochemistry ,Biophysics ,Steroid Isomerases ,Isomerase ,Biochemistry ,Catalysis ,Analytical Chemistry ,Enzyme catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Methionine ,Ketosteroid ,Catalytic Domain ,Organic chemistry ,Transition Temperature ,Enzyme kinetics ,Amino Acid Sequence ,Molecular Biology ,Alanine ,030102 biochemistry & molecular biology ,biology ,Hydrogen bond ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,biology.organism_classification ,Ketosteroids ,030104 developmental biology ,chemistry ,Amino Acid Substitution ,Mutation ,biology.protein ,Hydrophobic and Hydrophilic Interactions ,Sequence Alignment - Abstract
Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.
- Published
- 2016
4. Rapid Mapping of Active Site of KSI by Paramagnetic NMR
- Author
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Hyeong Ju Lee, Kwan Yong Choi, Hyung Jin Cha, Yong Nam Joe, and Hee Cheon Lee
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biology ,Stereochemistry ,Active site ,Substrate (chemistry) ,General Chemistry ,Crystal structure ,Isomerase ,chemistry.chemical_compound ,chemistry ,Reagent ,Ketosteroid ,medicine ,biology.protein ,Equilenin ,Heteronuclear single quantum coherence spectroscopy ,medicine.drug - Abstract
N HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediateanalog (equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active siteregion, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while theyexperienced full recovery of a lineshape upon the addition of equilenin. The mapped region was very similarto the active site of KSI as described by the crystal structure. These observations indicate that a combined useof paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site ofKSI, and can be applied to the analysis of both active site and function in unknown protein.Key Words : NMR, Ketosteroid isomerase, Active site, Paramagnetic agent, EquileninIntroductionIt has been known that the protein active site plays asignificant role in various physiological processes. Thecatalytic process is a binding reaction of protein similar to alock and key model where the substrate behaves like a keyfitting in the lock by interaction between substrate andresidues of active site in the protein. The active site of theprotein fits to the substrate conformation in size, shape,charge, and hydrophobic or hydrophilic character, or a littleconformational change is induced in order to ensure precisebinding to the substrate.
- Published
- 2012
5. Contribution of a low-barrier hydrogen bond to catalysis is not significant in ketosteroid isomerase
- Author
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Sejeong Shin, Do Soo Jang, Gildon Choi, Hyeong Ju Lee, and Hee Cheon Lee
- Subjects
Models, Molecular ,low-barrier hydrogen bond ,Stereochemistry ,Proton Magnetic Resonance Spectroscopy ,Low-barrier hydrogen bond ,Steroid Isomerases ,Isomerase ,Reaction intermediate ,Crystallography, X-Ray ,enzyme catalysis ,Article ,Catalysis ,Enzyme catalysis ,Substrate Specificity ,chemistry.chemical_compound ,Bacterial Proteins ,Ketosteroid ,Catalytic Domain ,Molecular Biology ,Equilenin ,Tyr14 ,biology ,Hydrogen bond ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,Cell Biology ,General Medicine ,chemistry ,Biochemistry ,Mutation ,biology.protein ,Biocatalysis ,ketosteroid isomerase ,Protein Binding - Abstract
Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.
- Published
- 2014
6. NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea
- Author
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Do Soo Jang, Hye Seon Moon, Hee Cheon Lee, Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, and Hyeong Ju Lee
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Models, Molecular ,Protein Denaturation ,Protein Folding ,biology ,Equilibrium unfolding ,Steroid Isomerases ,General Medicine ,Isomerase ,biology.organism_classification ,Biochemistry ,Pseudomonas putida ,Crystallography ,chemistry.chemical_compound ,chemistry ,Ketosteroid ,Urea ,Organic chemistry ,Protein folding ,Molecular Biology ,Protein secondary structure ,Dimerization ,Nuclear Magnetic Resonance, Biomolecular ,Heteronuclear single quantum coherence spectroscopy - Abstract
Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-(15)N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta6-strand. The results thus suggest that the interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.
- Published
- 2008
7. 15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility
- Author
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Do Soo Jang, Chul Hoon Kim, Ye Jeong Yoon, Hee Cheon Lee, and Hyeong Ju Lee
- Subjects
Models, Molecular ,Stereochemistry ,Mutant ,Steroid Isomerases ,Isomerase ,Biochemistry ,chemistry.chemical_compound ,Bacterial Proteins ,Ketosteroid ,Nandrolone ,Comamonas testosteroni ,Binding site ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Binding Sites ,biology ,Nitrogen Isotopes ,Chemistry ,Hydrogen bond ,Active site ,Hydrogen Bonding ,General Medicine ,biology.organism_classification ,Enzyme ,Amino Acid Substitution ,Mutation ,biology.protein ,Tyrosine - Abstract
The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.
- Published
- 2008
8. Alteration of the substrate specificity of ketosteroid isomerase from Pseudomonas putida Biotype B
- Author
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Kwan Yong Choi, Hyung Jin Cha, Do Soo Jang, and Han Seop Shin
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biology ,Isomerase ,biology.organism_classification ,Biochemistry ,Pseudomonas putida ,Microbiology ,chemistry.chemical_compound ,chemistry ,Ketosteroid ,Genetics ,Substrate specificity ,Molecular Biology ,Biotechnology - Published
- 2008
9. Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed 1H-15N multidimensional NMR spectroscopy
- Author
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Do Soo Jang, Hyeong Ju Lee, Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Hye Seon Moon, and Hee Cheon Lee
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Models, Molecular ,Protein Denaturation ,Protein Folding ,Magnetic Resonance Spectroscopy ,Xenon ,Chemistry ,Protein Conformation ,Pseudomonas putida ,Dimer ,Equilibrium unfolding ,Steroid Isomerases ,Nuclear magnetic resonance spectroscopy ,Isomerase ,Biochemistry ,Dissociation (chemistry) ,chemistry.chemical_compound ,Crystallography ,Kinetics ,Protein structure ,Ketosteroid ,Urea ,Protein folding ,Spectroscopy - Abstract
We used xenon-perturbed 1H-15N multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the beta3-, beta5- and beta6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level.
- Published
- 2007
10. A triple mutant (Y14F/Y30F/Y55F) of ketosteroid isomerase is more stable than its wild type through hydrophobic interaction as well as aromatic‐aromatic interaction
- Author
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Kwan Yong Choi, Hyung Jin Cha, Do Soo Jang, and Han Seop Shin
- Subjects
Hydrophobic effect ,chemistry.chemical_compound ,Biochemistry ,Chemistry ,Ketosteroid ,Genetics ,Wild type ,Isomerase ,Molecular Biology ,Biotechnology ,Triple mutant - Published
- 2007
11. Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B
- Author
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Bee Hak Hong, Kwan Yong Choi, Hyung Jin Cha, Heung-Soo Lee, Nam-Chul Ha, Do Soo Jang, Ja Young Lee, Byung-Ha Oh, and Sun Shin Cha
- Subjects
Models, Molecular ,Protein Denaturation ,Stereochemistry ,Mutant ,Steroid Isomerases ,Isomerase ,Biochemistry ,Catalysis ,Mutant protein ,Molecular Biology ,Binding Sites ,biology ,Hydrogen bond ,Chemistry ,Pseudomonas putida ,Active site ,Hydrogen Bonding ,Cell Biology ,biology.organism_classification ,Recombinant Proteins ,Kinetics ,Mutation ,biology.protein ,Thermodynamics ,Crystallization ,Isomerization ,Research Article - Abstract
KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.
- Published
- 2004
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