Structure Determination of the DHEThDP Intermediate in EcTK and hTK After numerous trials we succeeded in determination of x-ray structures with an acceptably high

occupancy of DHEThDP intermediate for hTK and EcTK (Fig. 53, Fig. 84). Notably, the condition in solution couldn´t been easily transferred to the crystalline state. For hTK we found that approx. 40-50 % DHEThDP intermediate had accumulated upon soaking with 20 mM for 3 min, whereas the remaining active sites contain just unsubstituted ThDP. Unfortunatly, the resolution of hTK crystals soaked with HPA didn´t exceed 1.45-1.6 Å resolution in numerous further trials. For EcTK we succeded in determination of two real atomic x-ray structures to 0.95 Å (5 min soak) and 0.97 Å (20 min soak) resolution after soaking in 20 mM HPA. Since the occupancy of the intermediate in the x-ray structure determined to 0.97 Å of 75 % (Rwork = 9.02, Rfree = 11.31, Fo > 4σ) was slightly better than for the second structure (0.95 Å, Rwork = 10.21, Rfree = 11.24, ≈ 50 % DHEThDP, data not shown) the discussion is focused on the slightly lower resolved complex. Importantly, even at very low contour levels in 2mFo-DFc (0.3-1σ) and mFo-DFc (1-3σ) electron density maps no traces of carboligation adducts are visible for all determined structures. Furthermore, because acetyl-ThDP (Fig. 51 b.)) couldn´t been detected under any chosen condition using NMR the trapped intermediate can be assigned as DHEThDP or the C2α-protonated form thereof. Unfortunately, a partial accumulation of the later form can neither be excluded by our x-ray crystallography results nor by the NMR-based intermediate method.

A fully unrestrained refinement was carried out for the model of the EcTK-DHEThDP complex using SHELXL-11 (Sheldrick, 2008). The outstanding resolution of the structure further enabled estimation of standard deviations for bond lengths. Because of the low occupancy in combination with

t (s)

-200 0 200 400 600 800 1000 1200

absorbance at 300 nm

108

the significantly lower resolution of 1.45 Å a refinement with weaker restrains for the hTK-DHEThDP intermediate molecule using PHENIX was not successful and caused disruption of the intermediate model. Thus, tighter refinement restrains had to be used in order to obtain a reliable model. The refined bond parameters for this structure are in consequence biased by the input restrains.

A detailed analysis of bond lengths for DHEThDP bound to EcTK (Tab. 13, Fig. 53) revealed that the C2-C2α bond (140 ± 2.7 pm) is much shorter than a standard C-C single bond (154 pm) consequently implying strong double character (typical C-C double bond 134 pm). In addition the C2α-O2α bond (123 ± 2.2 pm) has double bond character (143 pm for C-O single bond and 124 pm for C-O double bond). An analogous tendency for bond length is also observable for the intermediate in hTK (Fig. 84) and the second EcTK-DHEThDP complex (0.95 Å, data not shown).

Fig. 53: Detailed view on DHEThDP intermediate in EcTK (0.97 Å). On the left side the DHEThDP intermediate is shown surrounded by a 2mFo-DFc map contoured at 2σ (blue). DHEThDP and unreacted coenzyme are labeled.

Thiazolium moiety (top right) and aminopyrimidine moiety (bottom right) are shown with auxillary planes to illustrate deviations from planarity. The angular distortion (α) of the carbon bond connecting sugar and cofactor moiety of each intermediate is indicated (red). Selected atoms and distances (in pm) are labeled.

A very surprising and unexpected feature of the thiazolium ring is the significant out-of-plane distortion of C2 as well as of the C2-C2α bond. Contrary to the generally accepted, planar enamine state of DHEThDP in TK (Fiedler et al. 2002) we observe a considerable deviation from planarity of 14° (hTK) and 9.7° (EcTK) for the C2-C2α bond connecting cofactor and dihydroxyethyl moiety.

Although most of the bond lengths of the thiazol ring are suggestive for a predominantly aromatic character the C2-S1 bond (184.7 ± 2.2 pm) is drastically elongated, typical for a C-S single bond (Allen et al., 1987), which should result in reduction of aromaticity.

α= 9.7°

C2α

140 ± 2.7

C2´-M

N4´

C7´

123 ± 2.2

O2α

C2 C3α

O3α

C4

C5 C4-M

S1

N3

N1´

C2´ N3´

C4´

C5´

C6´

C7´

DHEThDP

unreacted ThDP

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Furthermore, the six-membered ring of the intermediate exhibits deviations from planarity for C2´, C7´and C2´-M but not for C4´and N4´. For the donor-ThDP intermediates a strong deviation from planarity for this entire ring and especially for C4´and N4´ was detectable suggesting that strain in this ring is partially released upon donor cleavage. However, a correlation of this structural change with catalytic competence must yet remain open.

A closer inspection of the intermediate revealed that C2α is neither trigonal planar, as it would be assumed for a sp² hybridized state, nor perfectly tetragonal (sp³ hybridized) in a protonated or carbanion state. Additionally, the thiazol ring nitrogen (N3) can serve as a monitor for the electron distribution of the intermediate. In the carbanion state N3 is planar whereas in an enamine or enolate state N3 adopts trigonal pyramidal geometry. The DHEThDP intermediate bound to EcTK possess a deviation of 16.5° from planarity at N3 but no perfect, trigonal pyramidal angles further suggesting that the electronic state of the intermediate can be explained with a more enamine-like structure.

By superposition of the post-cleavage intermediate in both transketolases structural differences for the thiazolium ring as well as for the diphosphate anchor are observable Fig.54. Additionally, we detect for EcTK a more pronounced deviation from planarity at C2α suggesting a higher fraction of C2α carbanion or C2α-protonated form for this TK.

Fig.54: Superposition of DHEThDP intermediates trapped in hTK and EcTK. In order to visualize structural changes between the DHEThDP intermediates trapped in the active sites of hTK (orange, 1.45 Å) and EcTK (blue, 0.97 Å) both structure are superimposed. Aminopyrimidine (AP ring) and thiazol ring (TH ring) as well as the diphosphate moiety (PP anchor) are labeled. Please note that TH ring and PP anchor adopt slightly different orientations whereas the AP ring is perfectly superimposable between both molecules. The substrate derived dihydroxyethyl moieties reveal small positional changes (top, zoom in).

zoom in C3α C2α

O2α

O3α

AP ring TH ring

PP anchor

110

Furthermore, both TK-DHEThDP complexes reveal the presence of a considerable fraction of un-reacted coenzyme. While for hTK this un-un-reacted fraction is perfectly superimposable with the coenzyme-derived part of the intermediate larger structural differences are observable for reacted and unreacted cofactor in EcTK. Similar structural differences were reported for the donor-ThDP intermediates in EcTK (Asztalos et al., 2007) relative to the ground state. These positional changes are locatable to the thiazolium ring, the diphosphate moiety and both methylene groups connecting the aforementioned moieties of the cofactor.