Hyungdon Yun

Konkuk University, Sŏul, Seoul, South Korea

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Publications (48)145.73 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k cat/K m value of 120.3 ± 11.5 [10(3) M(-1) s(-1)], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3 ± 1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.
    Applied Microbiology and Biotechnology 04/2014; · 3.69 Impact Factor
  • Advanced Synthesis & Catalysis 03/2014; · 5.54 Impact Factor
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    ABSTRACT: Here we enhanced the stability and biophysical properties of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. The global replacement of proline residue with (2S, 4R)-4-fluoroproline [(4R)-FP] into mRFP1 led to soluble protein but lost its fluorescence, whereas (2S, 4S)-4-fluoroproline [(4S)-FP] incorporation resulted in insoluble protein. The bioinformatics analysis revealed that (4R)-FP incorporation at Pro63 caused fluorescence loss due to the steric hindrance of fluorine atom of (4R)-FP with the chromophore. Therefore, Pro63 residue was mutated with the smallest amino acid Ala to maintain non coplanar conformation of the chromophore and helps to retain its fluorescence with (4R)-FP incorporation. The incorporation of (4R)-FP into mRFP1-P63A showed about 2∼3 fold enhancement in thermal and chemical stability. The rate of maturation is also greatly accelerated over the presence of (4R)-FP into mRFP1-P63A. Our study showed that a successful enhancement in the biophysical property of mRFP1-P63A[(4R)-FP] using non-canonical amino acid mutagenesis after mutating non-permissive site Pro63 into Ala.
    Biochemical and Biophysical Research Communications 09/2013; · 2.41 Impact Factor
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    ABSTRACT: In this study, we developed a one-pot one-step deracemization method for the production of various enantiomerically pure amines using two opposite enantioselective ω-TAs. Using this method, various aromatic amines were successfully converted to their (R)-forms (>99%) with good conversion.
    Chemical Communications 06/2013; · 6.38 Impact Factor
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    ABSTRACT: A current challenge in high-throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid-agar plate-based HTS assay for screening ortho-specific hydroxylation of daidzein by sensing formaldehyde generated from the O-dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde-specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho-carbon position to COH correlates with a linear relationship to O-dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4',7-dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B-ring, was examined for O-dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O-dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A-ring inactivated in order to find enhanced 3'-regioselectiviy). The whole-cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L(-1) production titer and greatly increased 3'-regioselectiviy (3'/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.
    ChemBioChem 06/2013; · 3.74 Impact Factor
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    ABSTRACT: Recently, ω-transaminases have been increasingly used to synthesize amine compounds by reductive amination of prochiral ketones which are of high pharmacological significance. However, the conventional methods for evaluating these enzymes are time consuming and have often been regarded as a bottle neck in developing these enzymes as industrial biocatalysts. In the past few years, several high throughput screening methods have been developed for fast evaluation and identification of ω-transaminase. This review summarizes the various methodologies developed for rapidly screening ω-transaminases.
    Biotechnology and Bioprocess Engineering 01/2013; 18:1-7. · 1.28 Impact Factor
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    ABSTRACT: Residue-specific incorporation is a technique used to replace natural amino acids with their close structural analogs, unnatural amino acids (UAAs), during protein synthesis. This is achieved by exploiting the substrate promiscuity of the wild type amino acyl tRNA synthetase (AARS) towards the close structural analogs of their cognate amino acids. In the past few decades, seleno-methionine was incorporated into proteins, using the substrate promiscuity of wild type AARSs, to resolve their crystal structures. Later, the incorporation of many UAAs showed that the AARSs are polyspecific to the close structural analogs of their cognate amino acids and that they maintain fidelity for the 19 natural amino acids. This polyspecificity helps to expand the use of this powerful tool to incorporate various UAA residues specifically through in vivo and in vitro approaches. Incorporation of UAAs is expensive, tedious and time-consuming. For the efficient incorporation of UAAs, it is important to screen substrate selectivity prior to their incorporation. As an initial study, using a docking tool, we analyzed the polyspecificity of the methionyl-tRNA synthetases (MetRSs) towards multiple reported and virtually generated methionine analogs. Based on the interaction result of these docking simulations, we predicted the substrate selectivity of the MetRS and the key residues responsible for the recognition of methionine analogs. Similarly, we compared the active site residues of the MetRSs of different species and identified the conserved amino acids in their active sites. Given the close similarity in the active site residues of these systems, we evaluated the polyspecificity of MetRSs.
    Journal of molecular graphics & modelling 11/2012; 39C:79-86. · 2.17 Impact Factor
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    ABSTRACT: Phenolic acid decarboxylase (PAD) catalyzes the non-oxidative decarboxylation of p-coumaric acid (pCA) to p-hydroxystyrene (pHS). PAD from Bacillus amyloliquefaciens (BAPAD), which showed k (cat)/K (m) value for pCA (9.3 × 10(3) mM(-1) s(-1)), was found as the most active one using the "Subgrouping Automata" program and by comparing enzyme activity. However, the production of pHS of recombinant Escherichia coli harboring BAPAD showed only a 22.7 % conversion yield due to product inhibition. Based on the partition coefficient of pHS and biocompatibility of the cell, 1-octanol was selected for the biphasic reaction. The conversion yield increased up to 98.0 % and 0.83 g/h/g DCW productivity was achieved at 100 mM pCA using equal volume of 1-octanol as an organic solvent. In the optimized biphasic reactor, using a three volume ratio of 1-octanol to phosphate buffer phase (50 mM, pH 7.0), the recombinant E. coli produced pHS with a 88.7 % conversion yield and 1.34 g/h/g DCW productivity at 300 mM pCA.
    Applied Microbiology and Biotechnology 10/2012; · 3.69 Impact Factor
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    ABSTRACT: A cytochrome P450 (CYP) enzyme, 3'-daidzein hydroxylase, CYP105D7 (3'-DH), responsible for daidzein hydroxylation at the 3'-position, was recently reported. CYP105D7 (3'-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3'-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3'-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3'-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.
    Microbial Cell Factories 06/2012; 11:81. · 3.31 Impact Factor
  • Sam Mathew, Hyungdon Yun
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    ABSTRACT: Transaminases have been increasingly used as efficient biocatalysts due to their ability to produce a wide range of optically pure amine compounds. Several approaches have been adopted, including screening, engineering, and develop-ment of new techniques in reaction systems for different aspects of the enzymes. This review summarizes the various methodologies and approaches adopted to produce enantio-merically pure amines and unnatural amino acids using ω-transaminases.
    ACS Catalysis 04/2012; 2(6):993-1001. · 5.27 Impact Factor
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    ABSTRACT: A deracemization method was developed to generate optically pure L-homoalanine from racemic homoalanine using D-amino acid oxidase and ω-transaminase. A whole cell reaction using a biphasic system converted 500 mM racemic homoalanine to 485 mM L-homoalanine (>99% ee).
    Organic & Biomolecular Chemistry 03/2012; 10(12):2482-5. · 3.57 Impact Factor
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    ABSTRACT: l-6-Hydroxynorleucine was synthesized from 2-keto-6-hydroxyhexanoic acid using branched-chain aminotransferase from Escherichia coli with l-glutamate as an amino donor. Since the branched-chain aminotransferase was severely inhibited by 2-ketoglutarate, the branched-chain aminotransferase reaction was coupled with aspartate aminotransferase and pyruvate decarboxylase. Aspartate aminotransferase converted the inhibitory 2-ketoglutarate back to l-glutamate by using l-aspartate as an amino donor. On the other hand, pyruvate decarboxylase further shifted the reaction equilibrium towards l-6-hydroxynorleucine through decarboxylation of pyruvate to acetaldehyde. The concerted action of the three enzymes significantly enhanced the yield compared to that of branched-chain aminotransferase alone. In the coupled reaction, 90.2 mM l-6-hydroxynorleucine (> 99% ee) was produced from 100 mM 2-keto-6-hydroxyhexanoic acid, whereas in a single branched-chain aminotransferase reaction only 22.5 mM l-6-hydroxynorleucine (> 99% ee) was produced.
    Biocatalysis and Biotransformation 03/2012; 30(2). · 0.90 Impact Factor
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    ABSTRACT: Recently, non-canonical amino acids (NCAA) incorporation was developed to enhance the functional properties of proteins. Incorporation of NCAA containing chlorine atom is conceptually an attractive approach to prepare pharmacologically active substances, which is a difficult task since chlorine is bulky atom. In this study, we evaluated the efficiency and extent of in vivo incorporation of tyrosine analogue 3-chlorotyrosine [(3-Cl)Tyr] into the recombinant proteins GFP and GFPHS (highly stable GFP). The incorporation of (3-Cl)Tyr into GFP leads to dramatic reduction in the expression level of protein. On the other hand, the incorporation of (3-Cl)Tyr into GFPHS was expressed well as a soluble form. In addition we used bioinformatics tools for the analysis to explore the possible constraints in micro-environment of each natural amino acid residue to be replaced with chlorine atom accommodation into GFPHS. In conclusion, our approaches are reliable and straightforward way to enhance the translation of chlorinated amino acids into proteins.
    Biotechnology and Bioprocess Engineering 03/2012; 17(6):679-. · 1.28 Impact Factor
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    ABSTRACT: CYP51, a sterol 14α-demethylase, is one of the key enzymes involved in sterol biosynthesis and requires electrons transferred from its redox partners. A unique CYP51 from Nocardia farcinica IFM10152 forms a distinct cluster with iron-sulfur containing NADPH-P450 reductase (FprD) downstream of CYP51. Previously, sequence alignment of nine reductases from N. farcinica revealed that FprC, FprD, and FprH have an additional sequence at their N-termini that has very high identity with iron-sulfur clustered ferredoxin G (FdxG). To construct an artificial self-sufficient cytochrome P450 monooxygenase (CYP) with only FprD, CYP51, and iron-sulfur containing FprD were fused together with designed linker sequences. CYP51-FprD fusion enzymes showed distinct spectral properties of both flavoprotein and CYP. CYP51-FprD F1 and F2 in recombinant Escherichia coli BL21(DE3) catalyzed demethylation of lanosterol more efficiently, with k(cat) /K(m) values of 96.91 and 105.79 nmol/min/nmol, respectively, which are about 35-fold higher compared to those of CYP51 and FprD alone.
    Biotechnology and Bioengineering 03/2012; 109(3):630-6. · 3.65 Impact Factor
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    ABSTRACT: A deracemization method was developed to generate optically pure L-homoalanine from racemic homoalanine using D-amino acid oxidase and ω-transaminase. A whole cell reaction using a biphasic system converted 500 mM racemic homoalanine to 485 mM L-homoalanine (>99% ee).
    Organic & Biomolecular Chemistry 01/2012; 10(12):2482-2485. · 3.57 Impact Factor
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    ABSTRACT: Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191-196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.
    PLoS ONE 01/2012; 7(12):e51510. · 3.73 Impact Factor
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    ABSTRACT: Among 33 cytochrome P450s (CYPs) of Streptomyces avermitilis, CYP102D1 encoded by the sav575 gene is naturally a unique and self-sufficient CYP. Since the native cyp102D1 gene could not be expressed well in Escherichia coli, its expression was attempted using codon-optimized synthetic DNA. The gene was successfully overexpressed and the recombinant CYP102D1 was functionally active, showing a Soret peak at 450 nm in the reduced CO difference spectrum. FMN/FAD isolated from the reductase domain showed the same fluorescence in thin layer chromatography separation as the authentic standards. Characterization of the substrate specificity of CYP102D1 based on NADPH oxidation rate revealed that it catalysed the oxidation of saturated and unsaturated fatty acids with very good regioselectivity, similar to other CYP102A families depending on NADPH supply. In particular, CYP102D1 catalysed the rapid oxidation of myristoleic acid with a k(cat)/K(m) value of 453.4 ± 181.5 μM(-1)·min(-1). Homology models of CYP102D1 based on other members of the CYP102A family allowed us to alter substrate specificity to aromatic compounds such as daidzein. Interestingly, replacement of F96V/M246I in the active site increased catalytic activity for daidzein with a k(cat)/K(m) value of 100.9 ± 10.4 μM(-1)·min(-1) (15-fold).
    FEBS Journal 12/2011; 279(9):1650-62. · 4.25 Impact Factor
  • Young-Man Seo, Hyungdon Yun
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    ABSTRACT: In this study, we demonstrated the asymmetric synthesis of L-tert-leucine from trimethylpyruvate using branchedchain aminotransferase (BCAT) from Escherichia coli in the presence of L-glutamate as an amino donor. Since BCAT was severely inhibited by 2-ketoglutarate, in order to overcome this here, we developed a BCAT/aspartate aminotransferase (AspAT) and BCAT/AspAT/pyruvate decarboxylase (PDC) coupling reaction. In the BCAT/ AspAT/PDC coupling reaction, 89.2 mM L-tert-leucine (ee >99%) was asymmetrically synthesized from 100 mM trimethylpyruvate.
    Journal of Microbiology and Biotechnology 10/2011; 21(10):1049-52. · 1.40 Impact Factor
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    ABSTRACT: Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagic Escherichia coli O157:H7. The antioxidant phloretin, which is abundant in apples, markedly reduced E. coli O157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensal E. coli K-12 biofilms. Also, phloretin reduced E. coli O157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyE and stx(2)), autoinducer-2 importer genes (lsrACDBF), curli genes (csgA and csgB), and dozens of prophage genes in E. coli O157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production in E. coli O157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory response in vitro using human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor of E. coli O157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensal E. coli biofilms.
    Infection and immunity 09/2011; 79(12):4819-27. · 4.21 Impact Factor
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    ABSTRACT: Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (-8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (-8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.
    Biotechnology Letters 07/2011; 33(11):2201-7. · 1.85 Impact Factor

Publication Stats

333 Citations
145.73 Total Impact Points

Institutions

  • 2014
    • Konkuk University
      • Department of Bioscience and Technology
      Sŏul, Seoul, South Korea
  • 2008–2013
    • Yeungnam University
      • School of Biotechnology
      Asan, South Chungcheong, South Korea
  • 2003–2013
    • Seoul National University
      • • Department of Chemical and Biological Engineering
      • • School of Chemical and Biological Engineering
      Seoul, Seoul, South Korea