Accumulation of the DHEThDP intermediate in the crystal structure of human TK with xylulose and fructose

The existence of DHEThDP intermediate has been textbook knowledge for years but remained elusive until Fiedler and colleagues (Fiedler et al., 2002) captured the three dimensional structure of this intermediate at the active site of yeast TK by cryocrystallography. The 1.9 Å crystal structure suggested that the C2-α carbanion/enamine intermediate existed as planar and E-configuration form. Later on in 2010 Suzuki and colleagues (Suzuki et al., 2010) observed the DHEThDP intermediate in phosphoketolase with a sp³ hybridized C2 atom which indicates non-planarity of the DHE moiety. Our previous colleagues confirmed the non-planarity of this intermediate by resolving sub-angstrom resolution crystal structures of EcTK socked with artificial substrate hydroxypyruvate (HPA) (Lüdtke, 2012). Since the DHEThDP intermediate is crucial in the thiamine catalysis, in this thesis we have identified a new route to capture this pivotal intermediate from non-phosphorylated sugars xylulose and fructose.

In addition, previous studies have shown that HPA is a suitable ketol donor substrate used to trap this particular intermediate DHEThDP. HPA first binds to the C2 position of ThDP and forms a short-lived intermediate hydroxylactyl-ThDP which undergoes decarboxylation to form DHEThDP by concomitant release of carbon dioxide as a by-product. Due to the virtue of the irreversibility of decarboxylation reaction, HPA is generally used for the asymmetric carbon-carbon formation in organic synthesis (Hailes et al., 2013). HPA is commercially

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available and relatively cheap, but it would not do any harm if a much cheaper ketol donor substituent could be found.

In this study, the accumulation of the post-cleavage DHEThDP intermediate using xylulose and fructose could be confirmed first by an acid quench/1H NMR measurement (Fig 33).

Comparison of non-phosphorylated sugars with HPA, some advantages are obviously seen.

First, the carbanion/enamine intermediate obtained from fructose/xylulose seems to be more stable than that from HPA which only has a half-time of 120 s^- (Lüdtke, 2012). Secondly, using of HPA would generate some other intermediates such as erythrulose-ThDP due to several off-pathway reactions (Asztalos, 2008), while the non-phosphorylated sugars only induce the formation of the desired intermediates. Thirdly, for biosynthetic purposes the availability of the six-carbon sugar fructose proves itself a much cheaper donor substrate for TKs relative to that of HPA. Although this combined acid quench/1H NMR method is very powerful in the analysis of different intermediates distribution of ThDP-dependent enzymes, it comes with limitation. Quench solution with strong acidity (pH 0.75) will render all compounds into conjugated acid which does not permit the resolution of their protonation states. Therefore, the carbanion, enamine or protonated form of DHEThDP can not be discriminated by this approach (Tittmann et al., 2003).

In the next step, the crystal structures of hTK with non-phosphorylated sugars xylulose and fructose are determined to medium resolution (~ 1.5 Å). For both structures, the electron density at the C2 of the cofactor ThDP clearly indicates the formation of the DHEThDP intermediate with high occupancy (Fig 35, 62, 70-80 % and 60-70 % for xylulose and fructose, respectively). But there is one thing to consider at this point, acetyl-ThDP (AcThDP), the dehydration form of the DHEThDP intermediate in the pathway of phosphoketolase reaction, has a similar chemical structure to the enamine/carbanion intermediate (Suzuki et al., 2010).

Since it could not be detected by NMR intermediate measurement, the existence of the AcThDP form therefore could not ambiguously excluded by the X-ray crystallography data.

Closer inspection of the structures reveals that the C2-C2α bond connecting cofactor and the DHE moiety exhibits an out-of-plane distortion (Fig 35). This partially suggests a tetrahedral arrangement of the bonding at C2, leading to the loss of aromaticity of the thiazolium ring.

Closer inspection reveals that C2 atom exhibits deviation from the thiazolium plane, which further supports the nonaromatic feature of the thiazolium ring. This observation has revised the knowledge that planar enamine state of DHEThDP intermediate exists in ScTK at a resolution of 1.9 Å (Fiedler et al., 2002). For the six-membered aminopyrimidine ring, deviations could also be observed for some atoms (Fig 35, N3’, C2’-M, C7’). Previous studies on pyruvate oxidase (POX) (Meyer et al., 2012) revealed that atom N4’ of the 4’-amino group

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deviates by approx. 10° from the aminopyrimidine plane, an interesting feature that is different from the enamine intermediate in hTK. This adopted high energy conformation is thought to facilitate the interconversion between various tautomeric states of the aminopyrimidine.

Therefore, we could speculate the deviation of the aminopyrimidine ring in the hTK-DHEThDP intermediate also play similar role during catalysis but this hypothesis remains to be tested.

As mentioned above, the bond lengths suggest the double bond character of the C2-C2α bond thereof sp2 hybridization of C2α atom, while the out-of-plane features partially indicate a tetrahedral arrangement of the bonding with sp3 hybridization at C2α atom. To distinguish between these two states, the geometry of N3 is employed as reference for the electron distribution. Interatomic distance of 1.44 ± 0.02 Å between C2 and N3 indicates single bond character (1.31 Å for the C3-N3 double bond observed in small molecule structure of thiamine models). Based on this, a more enamine-like structure should fit better to the electronic state of the DHEThDP intermediate.

In addition, the two leaving groups which are glyceraldehyde from xylulose and D-erythrose from fructose are also captured within the crystal structures (Fig 37, 38). The aldehyde trioses or tetroses would form the hydrate form or dimerize in aqueous solutions, as studies have shown that D-glyceraldehyde and D-erythrose mainly consist of mixtures of the dimeric forms in the syrupy state (Serianni et al., 1979). But we only capture the monomeric aldehyde form of these sugars in the crystal structure, suggesting that the active site of enzyme could sequester water molecules outside the protein and prevent hydration reaction to take place. In addition, the microenvironment of the active site does not provide enough space to accommodate two molecules of the released aldehyde sugars to form the dimeric form.

In order to analyse the structural changes of the cofactor ThDP upon the formation of the covalent intermediate DHEThDP intermediate by using xylulose and fructose, structures of hTK soaked with both sugars are superimposed with holo-hTK (1.75 Å, PDB: 3MOS).

Differences for the thiazolium ring (TH) as well as for the aminopyrimidine ring (AP) are observable whereas the diphosphate anchor (PP) is perfectly superimposable between both molecules. Slightly pronounced deviation for the planarity of C2α atom for the structure soaked with fructose suggests a higher fraction of C2α carbanion for this structure.

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Fig 56: Superposition of the ThDP molecule in holo-hTK (red) and covalent reaction intermediates DHEThDP in hTK soaked with xylulose (left panel, yellow) and fructose (right panel, blue). In order to visualize structural changes upon the formation of the DHEThDP intermediates by using the 5-carbon sugar xylulose and 6-carbon sugar fructose, both structures are superimposed with holo hTK. All structures are represented in ball-stick model, aminopyrimidine (AP ring) and thiazolium (TH ring) as well as the diphosphate moiety (PP anchor) are labelled.

Furthermore, the overall structure of the polypeptide chain in these two structures are very similar to the structure of holo-hTK in the resting state (RMSD of 0.35 Å and 0.41 Å for the structures of xylulose and fructose respectively for the monomer), suggesting that the active site is poised to accept donor substrate and conformational changes are not necessary during the enzyme reaction. In addition, both structures were found to be present in the monoclinic space group P21 with two monomers per asymmetric unit. The DHEThDP intermediate are present in both active sites with similar occupancy, ruling out the previous proposals of half-of-the-sites reactivity in ThDP enyzmes (Frank et al., 2007a), at least, in human transketolase.