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.

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.

Overlay of sulfonylurea structures. The five yeast AHAS polypeptides (taken from complexes) were superimposed to reveal the relative orientations of the sulfonylurea herbicides.

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 llBiochem. J. (1998) 335 (653–661)

G249

Species Mutation A. thaliana Resistance References

Yeast G116(NS) G121 SU [20]

Yeast A117X A122 SU [20,21]

E. coli A26V A122 SU [21]

A. thaliana M124E M124 SU, IM [11]

A. thaliana M124I M124 IM [11]

A. thaliana P197S P197 SU, TP [22,23]

Yeast P192(AELQRSVWY) P197 SU [20,24]

Tobacco P196(QS) P197 SU, IM [18,25]

A. thaliana R199(AE) R199 IM [11]

Yeast A200(CDERTVWY) A205 SU [20]

Yeast K251(DENPT) K256 SU [20]

Yeast M354(CKV) M351 SU [20]

Yeast D379(EGNPSVW) D376 SU [20]

E. coli M460(AN) M570 SU [26]

Yeast V583(ACNY) V571 SU [20]

Yeast W586X W574 SU [20]

Oilseed rape

W557L W574 SU, IM, TP [27]

Tobacco W573L W574 SU [25]

Cocklebur W552L W574 SU, IM, TP, PB [10]

Cotton W563(CS) W574 SU, IM [28]

E. coli W464(AFLQY) W574 SU [26]

Yeast F590(CGLNR) F578 SU [20]

A. thaliana S653N S653 IM, PB [23,29,30]

Biochem. J. (1998) 333 (765–777)

Table : Herbicide Contact Residues of AHAS

residue conserveda   closest contactb

Gly116 21 C   3.27 N1'

Val191 21 C 1 3.53 C6

Pro192 10 C 3.71 O7B

Ala195 3 C 4.42 C5

Ala200 21 C 3.58 C5

Phe201 21 O 3.47 C6

Lys251 21 C 3.70 O7B

Met354 21 S 3.71 O4'

Asp379 21 O 3.30 C5

Arg380 21 NH1 2.98 O9

Met582 20 O 3.61 C7'

Val583 21 C   3.78 C7'

Trp586 20 C 2 3.33 N3'

M.tuberculosis N.tabacum A.thaliana E.coli S.cerevisae Herbicides resistance

G62 G118 G121 G25 G116 SU (3.27A°)

A63 A119 A122 A26V A117 SU

L65 M122 M124 M28 L119 SU, IM

V137 V193 V99M V191 SU (3.53A°)

G138 P194 P197 S100P P192 SU, IM, TP (3.71A°)

G140 R196 R199 P102 S194 IM

A146 A202 A205 A108V A200 SU(3.58A°)

K197 K253 K256 K159 K251 SU (3.7A°)

M292 M347 M351 M250 M354 SU (3.71A°)

D317 D372 D376 D274 D379 SU, TP

M512 M567 M570 M460 M582 SU(3.61A°)

V513 V568 V571 V461 V583 SU (3.78A°)

W516 W573 W574 W464 W586 SU (3.33A°)

F520 F575 F578 F468 F590 SU

A593 S650 S653 P536S G657 IM, PB

Multiple sequence alignment of 4 different sources of ilvB (AHAS)

CS +Yeast AHAS

CS + Yeast AHAS

TB + Yeast AHAS

CS +Yeast AHAS

SM +Yeast AHAS

MM +Yeast AHAS

Cofactor +P.putida BFDC

Protein conc. (vol) Total Unit Total protein Sp. Activity (U/mg)

WT AHAS

0.866mg/mL 1.86U 14.8mg 0.000365

E85A AHAS

2.15mg/mL

(27mL)

0.475U 43.66mg 0.000025

(0.068% of wt)

E85D AHAS

1.815mg/mL

(27mL)

0.293U 69.66mg 0.000042

(0.115% of wt)

E85Q AHAS

2.22mg/mL

(20mL)

0.165U 65.12mg 0.000028

(0.077% of wt)

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 H2S04

0.5% creatine

5% α-naphthol

ε=20000M-1cm-1 of unknown color complex of acetolactate.

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 MA1 A2 A3 A4

20Kd M BI AI insoluble Q1 Q3Q2 Q4FT

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 βME 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