Publications by authors named "Bora Oh"

18 Publications

  • Page 1 of 1

Integrated prognostic and histogenomic justification of stage-directed therapy for single large hepatocellular carcinoma: a Korean nationwide registry study.

Gut 2021 Sep 7. Epub 2021 Sep 7.

Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Songpa-gu, The Republic of Korea

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http://dx.doi.org/10.1136/gutjnl-2021-325844DOI Listing
September 2021

Comprehensive characterization of viral integrations and genomic aberrations in HBV-infected intrahepatic cholangiocarcinomas.

Hepatology 2021 Sep 3. Epub 2021 Sep 3.

Asan Liver Center, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.

Background And Aims: Despite the epidemiological association between intrahepatic cholangiocarcinoma (iCCA) and HBV infection, little is known about the relevant oncogenic effects. We sought to identify the landscape and mechanism of HBV integration, along with the genomic architecture of HBV-infected iCCA (HBV-iCCA) tumors.

Approach And Results: We profiled a cohort of 108 HBV-iCCAs using whole-genome sequencing, deep sequencing, and RNA sequencing, together with preconstructed data sets of HBV-infected HCC (HBV-HCC; n = 167) and combined hepatocellular cholangiocarcinoma (HBV-cHCC/CCA; n = 59), and conventional (n = 154) and fluke-related iCCAs (n = 16). Platforms based on primary iCCA cell lines to evaluate the functional effects of chimeric transcripts were also used. We found that HBV had inserted at multiple sites in the iCCA genomes in 45 (41.7%) of the tumors. Recurrent viral integration breakpoints were found at nine different sites. The most common insertional hotspot (7 tumors) was in the TERT (telomerase reverse transcriptase) promoter, where insertions and mutations (11 tumors) were mutually exclusive, and were accompanied by promoter hyperactivity. Recurrent HBV integration events (5 tumors) were also detected in FAT2 (FAT atypical cadherin 2), and were associated with enrichment of epithelial-mesenchymal transition-related genes. A distinctive intergenic insertion (chr9p21.3), between DMRTA1 (DMRT like family A1) and LINC01239 (long intergenic non-protein coding RNA 1239), had oncogenic effects through activation of the mammalian target of rapamycin (mTOR)/4EBP/S6K pathway. Regarding the mutational profiles of primary liver cancers, the overall landscape of HBV-iCCA was closer to that of nonviral conventional iCCA, than to HBV-HCC and HBV-cHCC/CCA.

Conclusions: Our findings provide insight into the behavior of iCCAs driven by various pathogenic mechanisms involving HBV integration events and associated genomic aberrations. This knowledge should be of use in managing HBV carriers.
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http://dx.doi.org/10.1002/hep.32135DOI Listing
September 2021

Prognostic Molecular Indices of Resectable Hepatocellular Carcinoma: Implications of S100P for Early Recurrence.

Ann Surg Oncol 2021 Oct 30;28(11):6466-6478. Epub 2021 Mar 30.

Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.

Background: Although hepatocellular carcinomas (HCCs) often recur in patients undergoing hepatectomy, there are no reliable biomarkers of this undesirable event. Recent RNA-based efforts have developed valuable genetic indices prognostic of cancer outcomes. We aimed to identify molecular predictors of early recurrence after resection of HCC, and reveal the genomolecular structure of the resected tumors.

Method: Based on the transcriptomic and genomic datasets of 206 HCC samples surgically resected in the Asan Medical Center (AMC), we performed a differential gene expression analysis to identify quantitative markers associated with early recurrence and used the unsupervised clustering method to classify genomolecular subtypes.

Results: Differential gene expression profiling revealed that S100P was the highest-ranked overexpressed gene in HCCs that recurred within 2 years of surgery. This trend was reproduced in immunohistochemical studies of the original cohort and an independent AMC cohort. S100P expression also independently predicted HCC-specific mortality post-resection (adjusted hazard ratio 1.09, 95% confidence interval 1.01-1.19; p = 0.042). Validation in a Chinese cohort and in in vitro experiments confirmed the prognostic value of S100P in HCC. We further identified five discrete molecular subtypes of HCC; a subtype with stem cell features ('AMC-C4') was associated with the worst prognosis, both in our series and another two Asian datasets, and S100P was most strongly upregulated in that subtype.

Conclusion: We identified a promising prognostic biomolecule, S100P, associated with early recurrence after HCC resection, and established the genomolecular architecture of tumors affecting clinical outcomes, particularly in Asian patients. These new insights into molecular mediators should contribute to effective care for affected patients.
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http://dx.doi.org/10.1245/s10434-021-09825-yDOI Listing
October 2021

The HSP90 inhibitor, NVP-AUY922, sensitizes KRAS-mutant non-small cell lung cancer with intrinsic resistance to MEK inhibitor, trametinib.

Cancer Lett 2016 Mar 23;372(1):75-81. Epub 2015 Dec 23.

Institute for Innovative Cancer Research, College of Medicine, Asan Medical Center, University of Ulsan, Seoul 138-736, Republic of Korea; Department of Oncology, Asan Medical Center, Seoul, Republic of Korea. Electronic address:

RAS-driven tumors are often difficult to treat with conventional therapies and therefore, novel treatment strategies are necessary. The present study describes a promising targeted therapeutic strategy against non-small cell lung cancer (NSCLC) harboring KRAS mutations, which has intrinsic resistance to MEK inhibition. Results showed that intrinsic resistance to MEK inhibition occurred via high AKT expression by PI3K activation as a bypass pathway. The HSP90 inhibitor AUY922 suppressed PI3K-AKT-mTOR and RAF-MEK-ERK, and rendered cells sensitive to trametinib (GSK1120212). Synergy from the combination of the two drugs was observed in only sub-therapeutic concentrations of either drug. Dual inhibition of the HSP90 and MEK signaling pathways with sub-therapeutic doses may represent a potent therapeutic strategy to treat KRAS-mutant NSCLC with intrinsic resistance to MEK inhibition and to resolve the toxicity observed upon dual inhibition of AKT and MEK at therapeutic doses in clinical trials.
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http://dx.doi.org/10.1016/j.canlet.2015.12.015DOI Listing
March 2016

Elevated telomerase activity in essential thrombocythemia with extreme thrombocytosis.

Clin Biochem 2014 Apr 17;47(6):389-92. Epub 2014 Jan 17.

Department of Tumor Biology, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea. Electronic address:

Introduction: We performed a comparative analysis of telomerase activity (TA) in patients with myeloproliferative neoplasm (MPN) and myelodysplastic syndrome (MDS). The relationships between TA and known prognostic factors were also analyzed.

Materials And Methods: A telomeric repeat amplification protocol was performed with bone marrow hematopoietic cells from 96 normal controls, 44 MPNs, and 40 MDSs.

Result: TA (measured as molecules/reaction) showed no correlation with age in the control group (R(2)=0.0057, p=0.464). MPN showed elevated TA compared with controls (15,537.57 vs. 7775.44, p=0.020). Patients with essential thrombocythemia showed markedly elevated TA (22,688.56, p=0.030), particularly in cases with extreme thrombocytosis versus those without extreme thrombocytosis (34,522.19 vs. 9375.71, p=0.041). MDS patients showed a TA value of 7578.50.

Conclusion: There was no association between age and TA in bone marrow hematopoietic cells. TA was elevated in MPN but borderline in MDS, probably because of differences in the nature of the diseases. Elevated TA in patients with essential thrombocythemia, especially those with extreme thrombocytosis, suggests that an anti-telomerase strategy could be beneficial in the prevention of thrombotic complications.
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http://dx.doi.org/10.1016/j.clinbiochem.2014.01.006DOI Listing
April 2014

Synergistic killing effect of imatinib and simvastatin on imatinib-resistant chronic myelogenous leukemia cells.

Anticancer Drugs 2013 Jan;24(1):20-31

Department of Molecular and Clinical Oncology, Seoul National University College of Medicine, Seoul, Republic of Korea.

The antiproliferative effect of simvastatin on tumor cells has been speculated to be by intracellular signal inhibition through 3-hydroxy-3-methylglutaryl acetyl coenzyme A reductase. We examined the killing effect of simvastatin on imatinib-sensitive and resistant chronic myelogenous leukemia (CML) cells (three kinds of CML cell lines representative of each hematopoietic lineage: K562, KCL22, and LAMA84) and T315I and E255K site-directed mutant cells (Ba/F3). The in-vivo effect of simvastatin was determined in K562-xenografted nude mice. Cotreatment with imatinib and simvastatin in imatinib-resistant CML cells showed a synergistic killing effect in K562-R, KCL22-R, LAMA84-R, and E255K mutant cells, but only an additive effect in the T315I mutant cell, although a single treatment of simvastatin strongly inhibited T315I mutant cells. Mechanisms of killing were an induction of apoptosis and cell cycle arrest, through inhibition of tyrosine phosphorylation, and activated STAT5 and STAT3. Simvastatin suppressed the growth of K562-transplanted tumors, and cotreatment with imatinib was more effective in reducing tumor size. Simvastatin also killed primary CD34 cells from patients with CML more efficiently, compared with CD34 CML cells. Our study shows a synergic effect of imatinib and simvastatin both in imatinib-sensitive and imatinib-resistant cells, but more effective synergism in resistant cells. On the basis of these findings, we suggest that a combination of simvastatin and imatinib may be a potential candidate for the treatment of imatinib-resistant CML.
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http://dx.doi.org/10.1097/CAD.0b013e32835a0fbdDOI Listing
January 2013

Downregulation of Mcl-1 by daunorubicin pretreatment reverses resistance of breast cancer cells to TNF-related apoptosis-inducing ligand.

Biochem Biophys Res Commun 2012 May 25;422(1):42-7. Epub 2012 Apr 25.

Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. However, tumor cells often develop resistance to TRAIL, limiting its therapeutic potential. To study the mechanism underlying TRAIL-resistance in breast cancer cells, we performed a high-throughput compound screen in MCF-7 cells. We identified daunorubicin as a potent sensitizer of TRAIL-induced apoptosis in MCF-7 cells. Daunorubicin in combination with subtoxic concentrations of recombinant human TRAIL induced massive apoptosis in MCF-7 cells. This combination was effective in TRAIL-resistant MDA-MB-231 and T47D breast cancer cells. By immunoblotting, we found that daunorubicin treatment induced loss of the anti-apoptotic protein, Mcl-1, in breast cancer cells. RNA interference experiments revealed that reduced expression of Mcl-1 sensitized MCF-7 cells to TRAIL. Together, these data suggest that Mcl-1 is a major contributor to TRAIL-resistance in breast cancer cells, and that reduction of Mcl-1 protein levels using DNA damaging agents is a promising approach for cancer therapy.
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http://dx.doi.org/10.1016/j.bbrc.2012.04.093DOI Listing
May 2012

Increased copy number of the interleukin-6 receptor gene is associated with adverse survival in multiple myeloma patients treated with autologous stem cell transplantation.

Biol Blood Marrow Transplant 2011 Jun 8;17(6):810-20. Epub 2011 Jan 8.

Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, South Korea.

Interleukin-6 (IL-6) is a potent pleiotropic cytokine that regulates plasma cell (PC) growth via the IL-6 receptor (IL-6R). We hypothesized that up-regulation of IL-6R in myeloma cells might confer the growth privilege to myeloma cells over bone marrow (BM) hematopoietic cells. We investigated the frequency and prognostic implication of increased copy number of the IL-6R gene by fluorescence in situ hybridization (FISH) in patients with newly diagnosed multiple myeloma (MM). One hundred two patients with newly diagnosed MM were enrolled. The FISH study for IL-6R was performed using a homemade bacterial artificial chromosome (BAC) probe for IL6R at chromosome 1q21. FISH signals were counted among BM plasma cells sorted by cytoplasmic immunoglobulin light chain staining (cIg FISH). The amplification of IL-6R was detected in 53/102 patients (52.0%). The 5-year overall survival (OS) rate of patients with IL-6R gene amplification was 41.3% versus 44.8% for those with a normal IL-6R (P = .425). In 44 patients treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT), patients with ≥3.1 copy numbers of IL-6R per PC showed adverse 5-year OS compared to those with <2.1 copies of IL-6R gene (44.4% versus 78.0%, P = .024). In multivariate analysis, the increase of IL-6R copy numbers (mean copy/PC ≥3.1) could be considered as an independent prognostic factor for MM patients who underwent ASCT. The gain of the IL-6R gene was frequent in myeloma, showing an association with adverse prognosis in myeloma patients treated with ASCT. These findings suggest the potential role of IL-6R in myeloma cell growth and therapeutic implications of the IL-6R blocker in the future.
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http://dx.doi.org/10.1016/j.bbmt.2011.01.002DOI Listing
June 2011

Cytogenetic features of 5q deletion and 5q- syndrome in myelodysplastic syndrome in Korea; marker chromosomes proved to be chromosome 5 with interstitial deletion by fluorescence in situ hybridization.

Cancer Genet Cytogenet 2010 Dec;203(2):193-202

Department of Laboratory Medicine, Seoul National University College of Medicine, 101 Daehakro, Jongno-gu, Seoul 110-744, Korea.

We characterized the cytogenetic changes and prognostic characteristics of 133 Korean patients with myelodysplastic syndrome (MDS), focusing on 5q- syndrome and MDS with chromosome abnormalities involving 5q deletion according to World Health Organization 2008 classification. In all patients, G banding and fluorescence in situ hybridization for 5q were performed, and in MDS patients with 5q deletion, the deleted region on chromosome 5 was mapped with fluorescence in situ hybridization for EGR1, CSF1R, and PDGFRB. The frequency of isolated del(5q) syndrome and 5q deletion was 2.2% (3 of 137 patients) and 15.3% (21 of 137 patients), respectively. International Prognostic Scoring System (IPSS) groups were low risk (5.8%), intermediate 1 (51.1%), intermediate 2 (27.8%), and high risk (15.3%). The patients with del(5q) were significantly older (62 years) and showed an unfavorable survival compared to patients without del(5q). Half (53%) of the patients with del(5q) also had complex chromosome abnormalities, including chromosome 7 abnormalities. Of the patients with del(5q), 93.3% were deleted for all three regions on 5q, compared to 66.7% of patients with isolated del(5q). Marker chromosomes proved to be chromosome 5 with interstitial deletion of q arm by fluorescence in situ hybridization in three patients. The biological characteristics of MDS in Korea seem to be markedly different from those of Caucasians, with Koreans having a younger age, lower frequencies of 5q- syndrome, higher frequencies of complex cytogenetic abnormalities including del(5q), and poorer prognosis. We infer that additional chromosome abnormalities contribute to the adverse prognostic impact in patients with del(5q).
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http://dx.doi.org/10.1016/j.cancergencyto.2010.08.007DOI Listing
December 2010

Concurrent p16 methylation pattern as an adverse prognostic factor in multiple myeloma: a methylation-specific polymerase chain reaction study using two different primer sets.

Ann Hematol 2011 Jan 19;90(1):73-9. Epub 2010 Aug 19.

Department of Laboratory Medicine, Chosun University College of Medicine, Gwangju, South Korea.

Disruption of cell cycle control genes, including p16, is known to contribute to the cancerogenesis of multiple myeloma (MM). We investigated the methylation status of p16 and its association with common cytogenetic changes, clinicolaboratory findings, and survival in MM. Methylation-specific polymerase chain reaction was performed in 99 newly diagnosed MM patients using two different sets of primers (p16M1 and p16M2). Four patterns of p16 promoter methylation were observed: (1) concurrent methylation of p16M1 and p16M2 (P1P2), 27.3%; (2) methylation of p16M1 alone (P1N2), 7.1%; (3) methylation of p16M2 alone (N1P2), 26.3%; and (4) no methylation (N1N2), 39.4%. Patients with p16P1P1 showed shorter survivals than those with the other methylation patterns (P1N2, N1P2, or N1N2; median survival, 12 vs. 43 months; P < 0.001), regardless of the treatment protocol. In a multivariate analysis, p16P1P2 was an independent prognostic factor of adverse outcome in MM. According to International Staging System (ISS), the study population could be divided into 21.2% (20/94) for stage I, 22.3% (21/94) for stage II, and 56.4% (53/94) for stage III (P = 0.003). ISS can divide patients into prognostic groups. Of note, in patients older than 60 years, ISS was not reflective of disease stage (P = 0.114). If p16P1P2 sets up as stage 4 of ISS, modified ISS could be a more reliable staging system irrespective of age in Korean MM patients (P = 0.003 and P = 0.004 in patients younger than 60 years and in patients older than 60 years, respectively). Our study suggests the potential use of p16 methylation status in predicting the outcome of MM patients and the applicability of demethylating agents in MM.
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http://dx.doi.org/10.1007/s00277-010-1043-9DOI Listing
January 2011

Homozygous deletion of CDKN2A (p16, p14) and CDKN2B (p15) genes is a poor prognostic factor in adult but not in childhood B-lineage acute lymphoblastic leukemia: a comparative deletion and hypermethylation study.

Cancer Genet Cytogenet 2009 Nov;195(1):59-65

Department of Laboratory Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Chongro-gu, Seoul 110-744, South Korea.

The biological behavior of childhood B-lineage acute lymphoblastic leukemia (B-ALL) is different from that of adults. We performed a comprehensive analysis of the deletion and the methylation profile of CDKN2A (hereafter identified separately as p16 and p14, for the different proteins encoded) and CDKN2B (hereafter p15) in 91 newly diagnosed B-ALL patients (61 children, 30 adults). The prognostic significance of the profiles of these genes and the association between alterations in these genes and known cytogenetic prognostic factors (BCR/ABL; ETV6/RUNX1, formerly TEL/AML1; MLL rearrangement; and ploidy changes of chromosomes) were also assessed. The prevalence of homozygous deletion, hemizygous deletion, and no deletion of the 9p21 region was 11.5%, 16.4%, and 72.1%, respectively, in children and 30.0%, 20.0%, and 50.0%, respectively, in adults; the higher incidence of homozygous deletion in adults was significant (P=0.029). Homozygous deletion was associated with poor overall survival in adults (P=0.019), but not in children. The incidence of promoter methylation of p16, p14, and p15 was 34.4%, 14.8%, and 34.4%, respectively, in children and 26.7%, 10.0%, and 40.0%, respectively, in adults, with no significant difference between the two groups. No significant association was observed between deletion and methylation or with known cytogenetic prognostic factors. The difference in incidence, distribution, and prognostic effect of homozygous deletion in children and adults may explain the prognostic disparity.
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http://dx.doi.org/10.1016/j.cancergencyto.2009.06.013DOI Listing
November 2009

p15INK4b methylation correlates with thrombocytopenia, blast percentage, and survival in myelodysplastic syndromes in a dose dependent manner: quantitation using pyrosequencing study.

Leuk Res 2010 Jun 25;34(6):718-22. Epub 2009 Sep 25.

Department of Laboratory Medicine, Seoul National University College of Medicine, Chongno-Gu, Seoul 110-744, South Korea.

We investigated how the quantity of p15INK4b methylation related to International Prognosic Scoring System variables and survival in 74 patients with de novo myelodysplastic syndrome (MDS). Pyrosequencing of 11 consecutive CpG sites of the p15INK4b promotor region was performed, with the extent of CpG cytosine methylation assessed in terms of methylation level (MtL). Patients with >5% bone marrow blasts had higher MtL than patients with <5% blasts (10.1% vs. 6.1%, p=0.030, respectively). Methylation was not associated with chromosomal aberrations. The MtL of patients with thrombocytopenia were higher than patients without thrombocytopenia (11.2% vs. 6.2%, p=0.036, respectively); they were higher in patients with cytopenias in > or =2 lineages than in patients with either unilineage or no cytopenia (9.8% vs. 4.1%, p=0.036, respectively). The survival of patients with >7% MtL was worse than patients with <7% MtL (p=0.031). Heavy p15INK4b methylation in MDS is associated with IPSS predictors of poor prognosis and adverse survival.
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http://dx.doi.org/10.1016/j.leukres.2009.09.007DOI Listing
June 2010

Natural polyphenols antagonize the antimyeloma activity of proteasome inhibitor bortezomib by direct chemical interaction.

Br J Haematol 2009 Aug 3;146(3):270-81. Epub 2009 Jun 3.

Department of Molecular and Clinical Oncology, Seoul National University College of Medicine, Seoul, Korea.

Bortezomib is a therapeutic proteasome inhibitor with antimyeloma activity and polyphenols are well known compounds that exert antiproliferative effects against tumuors. We attempted to co-treat myeloma cells with bortezomib and polyphenols, anticipating a synergistic effect. However, the anticancer activity of bortezomib was blocked by the polyphenols. The structural features of the polyphenols correlated strikingly with their antagonistic effect; in particular, the presence or absence of a vicinal diol moiety was the key element for effective blockage of the anticancer function of bortezomib. We speculated that the vicinal diols in the polyphenols interact with the boronic acid of bortezomib and convert the active triangular boronic acid of bortezomib to an inactive tetrahedral boronate, thus abolishing the antimyeloma activity of bortezomib. We confirmed this hypothesis by (11)B nuclear magnetic resonance spectroscopy and an in vitro assay on multiple myeloma (MM) cell lines and primary myeloma cells from patients. Based on these findings, restriction of the intake of natural polyphenols in foods or vitamin supplements during bortezomib treatment in MM patients should be considered.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07752.xDOI Listing
August 2009

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues.

Biochem Biophys Res Commun 2004 Jun;319(2):486-92

Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799, Republic of Korea.

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.
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http://dx.doi.org/10.1016/j.bbrc.2004.05.017DOI Listing
June 2004

A bound water molecule is crucial in initiating autocatalytic precursor activation in an N-terminal hydrolase.

J Biol Chem 2004 Jan 8;279(1):341-7. Epub 2003 Oct 8.

Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, 28 Yongon-Dong, Seoul 110-799, Korea.

Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an alpha-subunit, a spacer sequence, and a beta-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1beta onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1beta to generate a strong nucleophile and to direct the OG atom of the Ser-1beta to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme.
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http://dx.doi.org/10.1074/jbc.M309281200DOI Listing
January 2004

Modifying the oligomeric state of cyclic amidase and its effect on enzymatic catalysis.

Biochem Biophys Res Commun 2003 Oct;310(2):651-9

Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799, South Korea.

A group of cyclic amidases, including hydantoinase, allantoinase, dihydropyrimidinase, and dihydroorotase, catalyze the reversible hydrolysis of cyclic ureides, such as 5-monosubstituted hydantoins and dihydropyrimidines. These four enzymes carry hydrophobic patches to form dimers. With the exception of dihydroorotase, these enzymes are further dimerized to form tetramers by hydrophobic interactions. This leads us to speculate that the hydrophobic interaction domain may be a significant factor in the catalytic property of these oligomeric cyclic amidases, for which activities are not allosterically regulated. We generated a dimeric D-hydantoinase by mutating five residues in the hydrophobic alpha-helical interface of a tetramer and analyzed the kinetic properties of the dimeric form of D-hydantoinase. The specific activity of the dimeric D-hydantoinase corresponds to 5.3% of the activity of tetrameric D-hydantoinase. This low specific activity of the dimeric D-hydantoinase indicates that the dimeric interaction to form a tetramer has a significant effect on the catalytic activity of this non-allosteric tetramer.
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http://dx.doi.org/10.1016/j.bbrc.2003.09.056DOI Listing
October 2003

Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues.

Biochem Biophys Res Commun 2003 Oct;310(1):19-27

Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799, Republic of Korea.

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.
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http://dx.doi.org/10.1016/j.bbrc.2003.08.110DOI Listing
October 2003
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