• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.387-397
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• 2023

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

• Bulletin of the Korean Chemical Society
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• 제29권7호
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• pp.1311-1314
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• 2008

Bacillus halodurans O-methyltransferase (BhOMT) is an S-adenosylmethionine dependent methyltransferase. In our previous study, three dimensional structure of the BhOMT has been determined by comparative homology modeling and automated docking study showed that two hydroxyl groups at 3'- and 4'-position in Bring and structural rigidity of C-ring resulting from the double bond characters between C2 and C3 of flavonoid, were key factors for interaction with BhOMT. In the present study, BhOMT was cloned and expressed. Binding assay was performed on purified BhOMT using fluorescence experiments and binding affinity of luteolin, quercetin, fisetin, and myricetin were measured in the range of $10^7$. Fluorescence quenching experiments indicated that divalent cation plays a critical role on the metal-mediated electrostatic interactions between flavonoid and substrate binding site of BhOMT. Fluorescence study confirmed successfully the data obtained from the docking study and these results imply that hydroxyl group at 7-position of luteolin, quercetin, fisetin, and myricetin forms a stable hydrogen bonding with K211 and carboxyl oxygen of C-ring forms a stable hydrogen bonding with R170. Hydroxyl group at 3'-and 4'-position in the B-ring also has strong $Ca^{2+}$ mediated electrostatic interactions with BhOMT.

• 치위생과학회지
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• 제2권2호
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• pp.121-124
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• 2002

본 연구에서는 구강미생물에서 발견되는 FTF 효소의 hydroxyapatite에 대한 친화도와 상호간의 결합에 영향을 주는 요소들을 비교하였으며, hydroxyapatite에 결합된 FTF 효소의 효소활성은 매우 안정적으로 높은 효소활성을 보였다. 결과를 요약하면 다음과 같다. 1. FTF는 치아를 대신하여 사용한 hydroxyapatite의 표면상태와 표면적에 영향을 받는 것으로 나타났다. 2. Hydroxyapatite에 결합된 FTF는 높은 플락탄 생성능을 보여, 비결합 FTF와 비교시, 약 70%의 효소활성을 나타내었다. 3. 결합된 FTF는 비결합 FTF와 비교시, 단백질분해효소의 분해작용에 대한 높은 저항성을 보여, hydroxyapatite에 결합된 효소에서는 3차원적인 구조적 변화가 나타나는 것으로 판단된다.

• Genomics & Informatics
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• 제19권3호
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• pp.29.1-29.10
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• 2021

In our previous studies, we have demonstrated the association of certain variants of the thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (TG) genes with congenital hypothyroidism. Herein, we explored the mechanistic basis for this association using different in silico tools. The mRNA 3'-untranslated region (3'-UTR) plays key roles in gene expression at the post-transcriptional level. In TSHR variants (rs2268477, rs7144481, and rs17630128), the binding affinity of microRNAs (miRs) (hsa-miR-154-5p, hsa-miR-376a-2-5p, hsa-miR-3935, hsa-miR-4280, and hsa-miR-6858-3p) to the 3'-UTR is disrupted, affecting post-transcriptional gene regulation. TPO and TG are the two key proteins necessary for the biosynthesis of thyroid hormones in the presence of iodide and H₂O₂. Reduced stability of these proteins leads to aberrant biosynthesis of thyroid hormones. Compared to the wild-type TPO protein, the p.S398T variant was found to exhibit less stability and significant rearrangements of intra-atomic bonds affecting the stoichiometry and substrate binding (binding energies, ΔG of wild-type vs. mutant: -15 vs. -13.8 kcal/mol; and dissociation constant, K_d of wild-type vs. mutant: 7.2E^-12 vs. 7.0E^-11 M). The missense mutations p.G653D and p.R1999W on the TG protein showed altered ΔG(0.24 kcal/mol and 0.79 kcal/mol, respectively). In conclusion, an in silico analysis of TSHR genetic variants in the 3'-UTR showed that they alter the binding affinities of different miRs. The TPO protein structure and mutant protein complex (p.S398T) are less stable, with potentially deleterious effects. A structural and energy analysis showed that TG mutations (p.G653D and p.R1999W) reduce the stability of the TG protein and affect its structure-functional relationship.

• Bulletin of the Korean Chemical Society
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• 제31권9호
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• pp.2497-2502
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• 2010

Arg13 is a conserved active-site residue in all known Pi class glutathione S-transferases (GSTs) and in most Alpha class GSTs. To evaluate its contribution to substrate binding and catalysis of this residue, three mutants (R13A, R13K, and R13L) were expressed in Escherichia coli and purified by GSH affinity chromatography. The substitutions of Arg13 significantly affected GSH-conjugation activity, while scarcely affecting glutathione peroxidase or steroid isomerase activities. Mutation of Arg13 into Ala largely reduced the GSH-conjugation activity by approximately 85 - 95%, whereas substitutions by Lys and Leu barely affected activity. These results suggest that, in the GSH-conjugation activity of hGST P1-1, the contribution of Arg13 toward catalytic activity is highly dependent on substrate specificities and the size of the side chain at position 13. From the kinetic parameters, introduction of larger side chains at position 13 results in stronger affinity (Leu > Lys, Arg > Ala) towards GSH. The substitutions of Arg13 with alanine and leucine significantly affected $k_{cat}$, whereas substitution with Lys was similar to that of the wild type, indicating the significance of a positively charged residue at position 13. From the plots of log ($k_{cat}/{K_m}^{CDNB}$) against pH, the $pK_a$ values of the thiol group of GSH bound in R13A, R13K, and R13L were estimated to be 1.8, 1.4, and 1.8 pK units higher than the $pK_a$ value of the wild-type enzyme, demonstrating the contribution of the Arg13 guanidinium group to the electrostatic field in the active site. From these results, we suggest that contribution of Arg13 in substrate binding is highly dependent on the nature of the electrophilic substrates, while in the catalytic mechanism, it stabilizes the GSH thiolate through hydrogen bonding.

• Molecules and Cells
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• 제41권4호
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• pp.331-341
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• 2018

L-pipecolic acid is a non-protein amino acid commonly found in plants, animals, and microorganisms. It is a well-known precursor to numerous microbial secondary metabolites and pharmaceuticals, including anticancer agents, immunosuppressants, and several antibiotics. Lysine cyclodeaminase (LCD) catalyzes ${\beta}$-deamination of L-lysine into L-pipecolic acid using ${\beta}$-nicotinamide adenine dinucleotide as a cofactor. Expression of a human homolog of LCD, ${\mu}$-crystallin, is elevated in prostate cancer patients. To understand the structural features and catalytic mechanisms of LCD, we determined the crystal structures of Streptomyces pristinaespiralis LCD (SpLCD) in (i) a binary complex with $NAD^+$, (ii) a ternary complex with $NAD^+$ and L-pipecolic acid, (iii) a ternary complex with $NAD^+$ and L-proline, and (iv) a ternary complex with $NAD^+$ and L-2,4-diamino butyric acid. The overall structure of SpLCD was similar to that of ornithine cyclodeaminase from Pseudomonas putida. In addition, SpLCD recognized L-lysine, L-ornithine, and L-2,4-diamino butyric acid despite differences in the active site, including differences in hydrogen bonding by Asp236, which corresponds with Asp228 from Pseudomonas putida ornithine cyclodeaminase. The substrate binding pocket of SpLCD allowed substrates smaller than lysine to bind, thus enabling binding to ornithine and L-2,4-diamino butyric acid. Our structural and biochemical data facilitate a detailed understanding of substrate and product recognition, thus providing evidence for a reaction mechanism for SpLCD. The proposed mechanism is unusual in that $NAD^+$ is initially converted into NADH and then reverted back into $NAD^+$ at a late stage of the reaction.

• 조명전기설비학회논문지
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• 제23권1호
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• pp.169-175
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• 2009

본 실험에서 자계중성방전 스파트링 시스템으로 균일한 산화 몰리브덴 박막을 얻을 수 있었다. 한편, 열처리 조건에 따라 박막의 제반특성은 XRD, XPS 및 SEM 등으로 고찰되었다. 기판의 열처리 온도에 따라 결정성장배향이 (100)에서 (210)으로 변함으로써, 박막의 결정성이 향상되었으며, 박막의 구조는 치밀해졌다. 광전자 Mo3d의 XPS 피크치는 결합에너지 228.9[eV]과 232.4[eV]에서 검출되었지만, O1s 피크치는 532.6[eV]였다. 서지 전압으로 방전시험은 연속적으로 10회 수행되었다. 전류-전압 특성곡선으로부터, 400[V]의 전압이 인가된 상태에서 시료의 초기 및 평균 저항치는 1.4[$M{\Omega}$]과 800[$M{\Omega}$]이었다.

• 한국미생물·생명공학회지
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• 제44권4호
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• pp.563-570
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• 2016

S. griseus wild type에서 dasA 유전자의 과발현에 의해 유도된 기저균사의 ectopic sporulation 관련 유전자를 알아보기 위해서, empty vector가 삽입된 균주와 dasA가 과발현된 균주의 전사체를 DNA microarray법으로 비교하였다. DNA microarray 결과를 토대로 dasA 유전자 과발현 균주에서 2배이상 발현량이 증가되었으며 p-value가 0.05 미만(p-value < 0.05)인 유전자들 중에서 false positive 를 제외시키는 작업을 통하여 최종적으로 4개의 유전자(SGR794, SGR2469, SGR3656, SGR3657)와 3개의 cluster (SGR795-797, SGR2377-2378, SGR6997-6998)를 선발하였다. 이들의 전사량은 low resolution Sl nuclease mapping 법을 통하여 dasA 유전자 과발현 균주에서 증가된 것을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1047-1053
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• 2005

Cyclosporin A binding protein type-19 kDa peptidyl-prolyl cis/trans isomerase (PPIases, EC 5.2.1.8) of Euglena gracilis was purified and some of its biochemical characters were elucidated. Purification of the PPIase was achieved by employing a series of steps involving ammonium sulfate precipitation, Superdex G-75 gel filtration chromatography, Mono­Q anion and Mono-S cation exchange chromatographies, and Superdex S-200 gel filtration chromatography on FPLC. Purified PPIase had a specific activity of 8,250 units/mg, showing a 27-fold increase compared with that of cell-free extract of Euglena gracilis. The enzyme consisted of a single polypeptide chain with a molecular mass of 19 kDa. It showed high substrate specificity to succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and $k_{car}/K_{m}$, for this substrate was found to be $61.19{\times}10^5/sec$. The isomer distributions were investigated at an equilibrium of seven different peptide substrates, varying Xaa in Suc-Ala-Xaa-Pro-Phe-p-nitroanilide in dimethylsulfoxide. The cis/trans equilibrium constants were estimated to be from 0.14 (Ile) to 0.63 (Gly), which correspond to $12.00\%\;to\;38.52\%$ of the cis population, respectively, under experimental condition. The enzyme was highly sensitive to the immunosuppressive ligand cyclosporin A, but not to other immunosuppressants such as FK506 and rapamycin. Thus, it appears to belong to the class of cyclophilin.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1636-1643
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• 2014

3-Hydroxybutyryl-CoA dehydrogenase is an enzyme that catalyzes the second step in the biosynthesis of n-butanol from acetyl-CoA, in which acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA. To understand the molecular mechanisms of n-butanol biosynthesis, we determined the crystal structure of 3-hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum (CbHBD). The monomer structure of CbHBD exhibits a two-domain topology, with N- and C-terminal domains, and the dimerization of the enzyme was mostly constituted at the C-terminal domain. The mode of cofactor binding to CbHBD was elucidated by determining the crystal structure of the enzyme in complex with $NAD^+$. We also determined the enzyme's structure in complex with its acetoacetyl-CoA substrate, revealing that the adenosine diphosphate moiety was not highly stabilized compared with the remainder of the acetoacetyl-CoA molecule. Using this structural information, we performed a series of site-directed mutagenesis experiments on the enzyme, such as changing residues located near the substrate-binding site, and finally developed a highly efficient CbHBD K50A/K54A/L232Y triple mutant enzyme that exhibited approximately 5-fold higher enzyme activity than did the wild type. The increased enzyme activity of the mutant was confirmed by enzyme kinetic measurements. The highly efficient mutant enzyme should be useful for increasing the production rate of n-butanol.