Jae-Woo Ahn

Kyungpook National University, Daikyū, Daegu, South Korea

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Publications (7)12.22 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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 of 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 of the production rate of n-butanol.
    Journal of microbiology and biotechnology. 08/2014;
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    ABSTRACT: ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as PHB, PHBHV, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that Km, Vmax, and Kcat values of 11.58μM, 1.5μmol/min, and 102.18 s(-1), respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.
    Biochemical and Biophysical Research Communications 01/2014; · 2.28 Impact Factor
  • Jae-Woo Ahn, Kyung-Jin Kim
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    ABSTRACT: Tuberculosis is a worldwide epidemic disease caused by Mycobacterium tuberculosis, with an estimated one-third of the human population currently affected. Treatment of this disease with aminoglycoside antibiotics has become less effective due to antibiotic resistance. Recent determination of the crystal structure of the M. tuberculosis Rv3168 protein suggests a structure similar to that of Enterococcus faecalis APH(3')-IIIa, and that this protein may be an aminoglycoside phosphotransferase. To determine whether Rv3168 confers antibiotic resistance against kanamycin, we performed dose response antibiotic resistance experiments using kanamycin. Expression of the Rv3168 protein in Escherichia coli conferred antibiotic resistance against 100 μM kanamycin, a concentration which effected cell growth arrest in the parental E. coli strain and an E. coli strain expressing the Rv3168 (D249A) mutant, in which the catalytic Asp249 residue was mutated to alanine. Furthermore, we detected a phosphotransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking-simulation of kanamycin into the Rv3168 structure suggests that kanamycin fits well into the substrate binding pocket of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position similar to that in E. faecalis APH(3')-IIIa. On the basis of these results, we suggest that the Rv3168 mediates kanamycin resistance in M. tuberculosis, likely through phosphotransferase targeting of kanamycin.
    Journal of Microbiology and Biotechnology 08/2013; · 1.40 Impact Factor
  • Proteins Structure Function and Bioinformatics 01/2012; 80(1):314-8. · 3.34 Impact Factor
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    ABSTRACT: Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD(+) rather than NADP(+) as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2 M calcium acetate, 0.1 M Tris-HCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The GluUADH crystal belonged to space group P6(3), with unit-cell parameters a = b = 122.58, c = 150.49 Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.78 Å(3) Da(-1). The structure was solved by the single anomalous dispersion method and structure refinement is in progress.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2011; 67(Pt 6):689-91. · 0.55 Impact Factor
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    ABSTRACT: Lysine biosynthesis has been of interest in plant research, because lysine is the most limiting amino acid in crop protein production. Dihydrodipicolinate synthase (DHDPS) catalyzes the branch point reaction leading to meso-diaminopimelate (DAP) and (S)-lysine in lysine biosynthesis. In this report, we present the crystal structure of DHDPS from the marine bacterium Hahella chejuensis (HcDHDPS) at 1.5 A resolution. The four subunits of the asymmetric unit assemble to form a tetramer with an approximate 222 symmetry. At the active site of HcDHDPS, three residues Tyr132, Thr43 and Tyr106 are observed to constitute a catalytic triad and are located at similar positions of the corresponding residues of Escherichia coli DHDPS. The structural similarities in the overall fold and the active site environment between these two enzymes imply that HcDHDPS functions by a mechanism similar to E. coli DHDPS. However, unlike E. coli DHDPS, HcDHDPS has a unique extensive dimer-dimer interface that is mediated by not only strong hydrophobic interactions but also a hydrogen bond network.
    International journal of biological macromolecules 03/2010; 46(5):512-6. · 2.37 Impact Factor
  • Jae-Woo Ahn, Yeon-Gil Kim, Kyung-Jin Kim
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    ABSTRACT: SSADH is involved in the final step of GABA degradation, converting SSA to succinic acid in the human mitochondrial matrix, and its activity is known to be regulated via 'redox-switch modulation' of the catalytic loop. We present the crystal structure of EcSSADH, revealing that the catalytic loop of EcSSADH, unlike that of human SSADH, does not undergo disulfide bond-mediated structural changes upon changes of environmental redox status. Subsequent redox change experiments using recombinant proteins confirm the non-redox regulation of this protein. Detailed structural analysis shows that a difference in the conformation of the connecting loop (beta15-beta16) causes the formation of a water molecule-mediated hydrogen bond network between the connecting loop and the catalytic loop in EcSSADH, making the catalytic loop of EcSSADH more rigid compared to that of human SSADH. The cytosolic localization of EcSSADH and the cellular function of the GABA shunt in E. coli might result in the non-redox mediated regulatory mechanisms of the protein.
    Biochemical and Biophysical Research Communications 01/2010; 392(1):106-11. · 2.28 Impact Factor

Publication Stats

15 Citations
12.22 Total Impact Points


  • 2010–2014
    • Kyungpook National University
      • School of Food Science and Biotechnology
      Daikyū, Daegu, South Korea
  • 2010–2012
    • Pohang University of Science and Technology
      • Pohang Accelerator Laboratory
      Andong, North Gyeongsang, South Korea