Structural and Functional Differences between human and E. coli Transketolase

HTK and EcTK reveal unique structural (Mitschke et al., 2010) and functional characteristics (Schenk et al., 1998). Since those differences are important for a mechanistic understanding the most important ones are described here.

Both TKs share a high amino acid sequence similarity of 48 % (26 % sequence identity). The monomers of hTK (pdb-code: 3MOS) and EcTK (pdb-code: 1QGD) can be superimposed with a root mean square deviation value of 2.3 Å and a high Dali Z score of 38.2 (586 C atoms were aligned) using the DaliLite v. 3 program (Holm and Rosenstrom, 2010) demonstrating a high structural similarity. While the overall fold of the dimeric enzyme and the topology of subunits or domains doesn´t deviate significantly between hTK and EcTK a remarkably structural difference is detectable for the linker region (Linker 1 in Fig. 13) between the PP and Pyr domain. Whereas this region is unstructured in hTK, the corresponding segment in EcTK is significantly longer (approx. 20 amino acids) and folds into a helix-turn-helix motif. Furthermore, several loops in hTK are shorter or completely missing which is a consequence of the smaller number of amino acid residues in hTK (623) in comparison to EcTK (663) (for detail see Fig.75).

Fig. 13: Superposition of hTK and EcTK monomers. The domain structure of bothTKs (EcTK grey, pdb code: 1QGD; hTK colored, pdb code: 3MOS) is shown in cartoon presentation with α-helices as barrels, β-sheets as arrows and loops or unstructured parts as wires.

The three domains of hTK are shown in different color code: N-terminal PP domain in green, middle PYR domain in orange and C-terminal domain in purple. Flexible linker of hTK are shown in red.

The active sites of both transketolases are well superimposable (Fig. 14). Aside from two amino acid exchanges all other residues necessary for substrate binding and catalysis are identical in both enzymes. Most importantly, a histidine residue (His473 in EcTK) that is strictly conserved in non-mammalian TKs is replaced by a glutamine (Gln428 in hTK) (Schenk et al., 1997). Hence, a function of this residue as acid/base catalyst in mammalian TKs has to be excluded (Schneider and Lindqvist, 1998). The second amino acid exchange is that of an unpolar

PP domain PYR domain C-terminal

domain

Linker 1 Linker 2

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isoleucine sitting atop of the cofactor thiazolium ring (Ile189 in EcTK) that is replaced by polar glutamine in hTK (Gln189). Because this residue is always a hydrophobic residue in non-mammalian TKs that spans into the substrate binding site suggest a function for Gln189 in substrate binding and orientation in reactant states (Mitschke et al., 2010).

Fig. 14: Superposition of the active sites of hTK and EcTK. Detailed view of the substrate binding pocket (cartoon presentation) onto the bound cofactor. For reasons of clarity just selected active site residues are shown and labeled (ball and stick representation, EcTK (yellow), hTK (purple)). Labels belonging to EcTK are underlined. ThDP bound to EcTK (red) and hTK (green) as well as the bivalent cation are labeled.

A yet undescribed structural difference between both TKs is the conformation of the bound coenzyme which is difficult to observe by over-all superposition of both structures but well visible by individual alignment of both ThDP molecules as shown in Fig.15. While both cofactors adopt the canonical V-like conformation and are well superimposable for the aminopyrimidine (AP) and thiazolium (TH) ring, a positional difference is observable for the diphosphate moiety which functions to bind the cofactor to the enzyme. Additionally, ThDP bound to EcTK adopts a more compact V-like conformation which is reflected by the small structural difference at the top of the AP ring.

Gln189

Gln428 His473 Ile189

Phe434 Phe389

Phe437 Phe392

His 77 His66

Glu411 Glu366

ThDP in EcTK

ThDP in hTK His261

His258

Ca²⁺/Mg²⁺

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Fig.15: Superpositions of active-site bound ThDP in EcTK (red) and hTK (green). Cofactors` aminopyrimidine- (AP ring) and thiazolium ring (TH ring) as well as the pyrophosphate moiety (PP anchor) are labeled.

For conversion of native substrates X5P and R5P EcTK has a kcat value of 40-50 s^-1 (Asztalos, 2007 b;

Sprenger et al., 1995) whereas kcat values reported for hTK are approx. 3-4fold lower (Mitschke et al., 2010; Schenk et al., 1998). Furthermore, Schenk and colleagues reported a norrower substrate specificity of hTK relative to EcTK (Schenk et al., 1998). Both kinetic differences are not yet understood.

Previous work (Mitschke, 2008) and initial kinetic and biophysical experiments revealed that human transketolase is prone to aggregation. Especially rapid mixing techniques, as well as working at moderate temperature (25 °C–37 °C) caused visible aggregation of the protein. Since protein aggregation is concentration dependent methods that require high protein concentrations are severely affected by this undesired site effect. To test if the presence of additives or/and cofactors have a positive effect on hTK stability/solubility a fluorescence based assay was performed. Here, we observe very similar apparent unfolding temperature in absence and presence (up to 700 mM) of different additives like NaCl, KCl, MgCl2, imidazol and ThDP (data not shown). Moreover, the thermodynamic stability of hTK represented by a moderate, apparent unfolding temperature of 70.5 °C (Fig. 16) can be excluded as source of precipitation. Remarkably, apo (56.4 °C) and holo (58.2 °C) EcTK have identical apparent unfolding temperatures, while apo hTK (55 °C) is thermodynamically unstable relative to holo hTK demonstrating a higher importance of cofactor binding for enzyme stability in hTK. These findings are in line with previous studies demonstrating that ThDP is irreversibly bound by hTK whereas EcTK binds the coenzyme reversibly (Schenk et al., 1998; Wang et al., 1997). The molecular origin of this difference is currently under investigation in our group. Since we have yet solely determined the structure of apo hTK (data not shown) a structural comparison of both apo TKs is not feasible.

While protein crystallization worked well for hTK, other techniques (fast kinetics, ITC) could not be performed with the human enzyme as aggregation falsifies a quantitative analysis or makes it even impossible. For this reason we performed these methods solely with EcTK. This TK offers additional advantages as it can be produced easily in very high amounts and stored for month without losing a considerably fraction of activity (Schenk et al., 1998).

AP ring TH ring

PP anchor

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Fig. 16: Thermodynamic stability of hTK and EcTK. Temperature-induced unfolding of of 100 µg/ml hTK or EcTK in 50 mM Na phosphate (pH 7.5), 2.5 mM MgCl2, in absence (apo) or presence (holo) of 300 µM ThDP. Data points were fitted (black lines) according to eq. 2 (Pace et al., 1998).