Role of hydrogen bond network in the function, stability, and structure of ketosteroid isomerase from Pseudomonas putida

차형진,장도수,김연길,최관용 한국구조생물학회 2015 Biodesign Vol.3 No.2

H-bond networks that are formed by polar or charged residues play a critical role in the structure, catalytic activity andstability of proteins. Ketosteroid isomerase (KSI) catalyzes the allylic isomerization of a variety of Δ5-3-ketosteroids by theintramolecular transfer of a proton from the 4β-position to the 6β-position via a dienolate intermediate. An H-bond networkin KSI consists of two catalytic residues (Tyr14 and Asp99), Tyr30, Tyr55, and a water molecule in the highly hydrophobicactive site. In this review, we summarize our current understanding of the role of the H-bond network in the activity,stability, and structure of KSI. Both mutational and structural studies suggest that the H-bond newtork could contributeto the catalysis and stability of KSI through the maintenance of the active-site geometry and the provision of structuralsupport.

Rescue of Deleterious Mutations by the Compensatory Y30F Mutation in Ketosteroid Isomerase

차형진,최관용,장도수,김연길,홍비학,우재성,김경태 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1

Proteins have evolved to compensate for detrimental mu-tations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isome-rase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Pre-vious 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.

Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

장도수,최길돈,차형진,신세정,홍비학,이희천,이형주,최관용 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.5

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5-3- ketosteroid to its conjugated Δ4-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.

Rapid Mapping of Active Site of KSI by Paramagnetic NMR

Yong Nam Joe,차형진,이형주,Kwan Yong Choi,Hee Cheon Lee 대한화학회 2012 Bulletin of the Korean Chemical Society Vol.33 No.9

Active site mapping has been done for Δ5-3-ketosteroid isomerase (KSI) by analyses of paramagnetic effect on 1H-15N HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediate analog (equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active site region, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while they experienced full recovery of a lineshape upon the addition of equilenin. The mapped region was very similar to the active site of KSI as described by the crystal structure. These observations indicate that a combined use of paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site of KSI, and can be applied to the analysis of both active site and function in unknown protein.

Apoptosis-related mRNA expression profiles of ovarian cancer cell lines following cisplatin treatment

윤주희,김응삼,이성종,박창욱,차형진,홍비학,최관용 대한부인종양학회 2010 Journal of Gynecologic Oncology Vol.21 No.4

Objective: The aim of this study was to identify apoptosis-related genes of ovarian cancer cell lines following cisplatin treatment. Methods: We used IC50 values and fluorescence-activated cell sorting analysis to compare cell death in 2 ovarian cancer cell lines, namely, SKOV-3 and OVCAR-3, upon treatment with cisplatin. Moreover, the change in transcriptional levels of apoptosis-associated genes was measured with a dendron-modified DNA microarray. Results: The protein levels for the up-regulated genes in each cell line were validated to identify the molecules that may determine the cellular behavior of cisplatin resistance. Eight genes were over-expressed in the 2 cell lines. The cisplatin-induced up-regulation of DAD1 in transcriptional and protein levels contributed to the cisplatin resistance of OVCAR-3, and the up-regulation of FASTK and TNFRSF11A in SKOV-3 resulted in its higher sensitivity to cisplatin than that of OVCAR-3. Conclusion: In the present study, we have identified a set of genes responsible for apoptosis following cisplatin treatment in ovarian cancer cell lines. These genes may give information about the understanding of cisplatin-induced apoptosis in ovarian cancer.

리샤주 운동을 이용한 팔레트 홀 인식 라이다 기구 설계

김경수(K. S. Kim),최경호(K. H. Choi),전원일(W. I. Jeon),손지민(J. M. Son),차형진(H. J. Cha),조방현(B. H. Cho),우상하(S. H. Woo),이대엽(D. Y. Lee) 유공압건설기계학회 2021 유공압건설기계학회 학술대회논문집 Vol.2021 No.7

Recognition of surrounding objects in industrial equipment, including unmanned forklifts, is a very important function. In order to recognize a two-dimensional object using a one-dimensional lidar, a study was conducted to develop a device that drives the lidar to recognize two-dimensional positions. In this study, a device that implements sine wave motion on two axes using two stepper motors was developed in order to recognize the features of surrounding objects by moving the one-dimensional lidar in a lissajous pattern. For this, a motion conversion mechanism was designed and applied so that the lidar can move with the required accuracy. Applying the results of this study to the device for recognizing the location of the hole in the pallet, it was confirmed that the shape and center of the hole can be recognized.