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20050801
 

20050801

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work on AHAS

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    20050801 20050801 Presentation Transcript

    • Inhibitor binding sites of M.tuberculosis AHAS
    • Structure of AHAS. Schematic representation of the structure of yeast AHAS in complex with CS. active site of AHAS
    • Structure of monomer B in the Yeast-CE complex (Pang et al. J Biol Chem 2003) a , structure of monomer B in the yeast AHAS·CE complex. The amino acid residues that are not observed in the structure of the free enzyme are colored green (the mobile loop) and brown (the C-terminal arm). ThDP and FAD are displayed as tan-colored ball-and-stick models . b , major differences in the structure of yeast AHAS in the presence and absence of CE. The mobile loop region, FAD, and ThDP are shown in green (with CE) and blue (without CE). The C-terminal arm and CE are shown in brown.
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    • Stereo diagram of the active site of AHAS II, based on the structure of the yeast AHASCIE complex (Protein Data Bank entry 1NOH) . The residues are labeled with the numbering of AHAS II, and residues belonging to the second catalytic polypeptide are marked with an asterisk . Thiamin diphosphate is shown with dark bonds , with its aminopyrimidine to the left and diphosphate to the right. C-2, the activated carbon, is labeled. Conformation A of Trp586 in Protein Data Bank entry 1N0H was used for Trp464.
    • Proposed structure for 2-ketobutyrate in the active site of the AHAS II-HEThDP intermediate. The angle of view is slightly different from that in Fig. 6, and peripheral residues have been eliminated so that the substrate can be visualized. A thin dotted red line connects the nucleophilic carbon of HEThDP and the carbonyl carbon of 2-ketobutyrate. Hydrogen bonds of interest are marked with dashed lines . B , schematic diagram with distances.
    • Stereo picture of the proposed structure for benzaldehyde in the active site of the AHAS II-HEThDP intermediate.
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    • Overlay of sulfonylurea structures. The five yeast AHAS polypeptides (taken from complexes) were superimposed to reveal the relative orientations of the sulfonylurea herbicides.
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    • herbicide binding site of AHAS. Distances in angstroms from CS are indicated with broken lines for hydrophobic contacts (black) and hydrogen bonds (blue). Distance cutoffs of 3.2 and 3.9 Å were employed for hydrogen bond and van der Waals interactions, respectively
    • MM + yeast
    • SM + yeast
    • TB + yeast
    • Herbicide binding sites of E.coli AHAS ll Biochem. J. (1998) 335 (653–661) G249
    • Biochem. J. (1998) 333 (765–777) [ 23 , 29 , 30 ] IM, PB S653 S653N A. thaliana [ 20 ] SU F578 F590(CGLNR) Yeast [ 26 ] SU W574 W464(AFLQY) E. coli [ 28 ] SU, IM W574 W563(CS) Cotton [ 10 ] SU, IM, TP, PB W574 W552L Cocklebur [ 25 ] SU W574 W573L Tobacco [ 27 ] SU, IM, TP W574 W557L Oilseed rape [ 20 ] SU W574 W586X Yeast [ 20 ] SU V571 V583(ACNY) Yeast [ 26 ] SU M570 M460(AN) E. coli [ 20 ] SU D376 D379(EGNPSVW) Yeast [ 20 ] SU M351 M354(CKV) Yeast [ 20 ] SU K256 K251(DENPT) Yeast [ 20 ] SU A205 A200(CDERTVWY) Yeast [ 11 ] IM R199 R199(AE) A. thaliana [ 18 , 25 ] SU, IM P197 P196(QS) Tobacco [ 20 , 24 ] SU P197 P192(AELQRSVWY) Yeast [ 22 , 23 ] SU, TP P197 P197S A. thaliana [ 11 ] IM M124 M124I A. thaliana [ 11 ] SU, IM M124 M124E A. thaliana [ 21 ] SU A122 A26V E. coli [ 20 , 21 ] SU A122 A117X Yeast [ 20 ] SU G121 G116(NS) Yeast References Resistance A. thaliana Mutation Species
    • Table : Herbicide Contact Residues of AHAS N3' 3.33 C 2 20 Trp586 C7' 3.78 C   21 Val583 C7' 3.61 O 20 Met582 O9 2.98 NH1 21 Arg380 C5 3.30 O 21 Asp379 O4' 3.71 S 21 Met354 O7B 3.70 C 21 Lys251 C6 3.47 O 21 Phe201 C5 3.58 C 21 Ala200 C5 4.42 C 3 Ala195 O7B 3.71 C 10 Pro192 C6 3.53 C 1 21 Val191 N1' 3.27 C   21 Gly116 closest contact b   conserved a residue
    • SU F590  F468 F578  F575 F520 SU (3.78A °) V583  V461 V571  V568 V513 SU(3.61A °) M582  M460  M570  M567 M512 P536 S W464  D274 M250 K159 A108 V  P102 S100 P V99 M  M28 A26 V  G25 E.coli SU (3.7A °) K251  K256  K253 K197 SU(3.58A °) A200  A205  A202 A146 SU (3.53A °) V191  V193 V137 SU, IM L119 M124  M122 L65 SU A117  A122  A119 A63 SU (3.71A °) M354  M351  M347  M292 IM S194 R199  R196 G140 SU, IM, TP (3.71A °) P192  P197  P194  G138 S650 W573  D372 G118 N.tabacum S653  W574  D376  G121  A.thaliana SU (3.27A °) G116 G62 Herbicides resistance S.cerevisae M.tuberculosis SU, TP D379  D317 IM, PB G657 A593 SU (3.33A °) W586  W516
    • Multiple sequence alignment of 4 different sources of ilvB (AHAS)
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    • CS +Yeast AHAS CS + Yeast AHAS
    • TB + Yeast AHAS
    • CS +Yeast AHAS SM +Yeast AHAS
    • MM +Yeast AHAS
    • Cofactor +P.putida BFDC
    • Activity Test: Reaction vol. 200mL Reaction Mixture: 100mM Pyruvate, 1mm ThDP, 10mM Mg++ and 50mM FAD incubate 60 min at 37℃ Reaction stopped by 8N H 2 S04 0.5% creatine 5% α -naphthol ε =20000M- 1 cm- 1 of unknown color complex of acetolactate. 0.000042 (0.115% of wt) 69.66 mg 0.293U E85D AHAS 1.815 mg/mL (27mL) 0.000025 (0.068% of wt) 43.66 mg 0.475U E85A AHAS 2.15 mg/mL (27mL) 0.000365 14.8 mg 1.86U WT AHAS 0.866 mg/mL 0.165U Total Unit 0.000028 (0.077% of wt) 65.12 mg E85Q AHAS 2.22 mg/mL (20mL) Sp. Activity (U/mg) Total protein Protein conc. (vol)
    • E85A LB A600=0.604; IPTG 0.1mM 12 hrs induction at 18oC E85Q LB A600=0.567; IPTG 0.1mM 18hrs induction at 18oC BI AI Insoluble resin 20Kd M A1 A2 A3 A4 20Kd M BI AI insoluble Q1 Q3 Q2 Q4 FT FT Wash
      • Result of this week:
      • To increase solubility Triton X-100 (0.5%) used while elution
      • In order to remove high conc. Of NaCl, and Imidazole salt, purified proteins are dialyzed against 50mM TRIS pH8.0, 5mM EDTA and 1mM β ME and concentrated by Millipore (10YT).
      • Using G-10 desalting column protein became salt free (20mM TRIS pH 8.0)
      • In order to remove Triton X-100 we used Q-sepharose anion exchange column (elution buffer 20mM TRIS and 1M NaCl pH 8.0)
      • Protein conc. determined by Bradford method and it might be interfered with Triton X-100.
      • Purified Protein checked by 12% SDS-PAGE