Journal of Molecular Biology
Regular ArticleRefined Structure of Transketolase from Saccharomyces cerevisiae at 2·0 Å Resolution
Abstract
The crystal structure of transketolase from Saccharomyces cerevisiae has been refined to a crystallographic residual of 15·7% at 2·0 Å resolution using the program package X-PLOR. The refined model of the transketolase homodimer, corresponding to 1356 amino acid residues in the asymmetric unit, consists of 10,396 protein atoms, 1040 solvent molecules, 52 thiamine diphosphate atoms and two calcium ions.
All amino acid residues except for the two N-terminal residues of the two subunits are defined in the electron density maps and refined. The estimated root-mean-square (r.m.s.) error of the model is less than 0·2 Å as deduced from Luzzati plots. The r.m.s. deviation from ideality is 0·017 Å for bond distances and 3·1° for bond angles. The main-chain torsion angles of non-glycine residues lie within the allowed regions of the Ramachandran plots. The model shows a very good fit to the electron density maps. The average B-factor for all protein atoms in the first subunit is 19Å2, and 15Å2 in the second. The average B -factor for solvent atoms is 32Å2.
The two subunits of transketolase were refined independently and have nearly identical structures with an r.m.s. deviation of 0·24Å for Cα atoms 3 to 680, and slightly less when aligning the individual domains. A few exceptions from the 2-fold symmetry are found, mostly in the surface residues.
The thiamine diphosphate cofactors have identical conformations. The cofactor is shielded from except for the C-2 atom of the thiazolium ring. A calcium ion is bound to the diphosphate group of thiamine and protein ligands. The metal binding site and the interactions of thiamine diphosphate with protein residues are described. A network of hydrogen bonds consisting of glutamic acid residues and internal water molecules connects the two thiamine diphosphate molecules. Its structure and possible functional implifications are discussed.
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Frontiers in the enzymology of thiamin diphosphate-dependent enzymes
2022, Current Opinion in Structural BiologyEnzymes that use thiamin diphosphate (ThDP), the biologically active derivative of vitamin B1, as a cofactor play important roles in cellular metabolism in all domains of life. The analysis of ThDP enzymes in the past decades have provided a general framework for our understanding of enzyme catalysis of this protein family. In this review, we will discuss recent advances in the field that include the observation of “unusual” reactions and reaction intermediates that highlight the chemical versatility of the thiamin cofactor. Further topics cover the structural basis of cooperativity of ThDP enzymes, novel insights into the mechanism and structure of selected enzymes, and the discovery of “superassemblies” as reported, for example, acetohydroxy acid synthase. Finally, we summarize recent findings in the structural organisation and mode of action of 2-keto acid dehydrogenase multienzyme complexes and discuss future directions of this exciting research field.
The mechanism of a one-substrate transketolase reaction. Part II
2021, Analytical BiochemistryCitation Excerpt :The dimer structure of TK is formed largely via the interaction between PP- and Pyr-domains. The coenzyme, ThDP, interacts with the same domains [6] (Scheme 1). TK has two active sites located in a deep cleft between the subunits, which determines their interaction.
In a recent paper, we showed the difference between the first stage of the one-substrate and the two-substrate transketolase reactions – the possibility of transfer of glycolaldehyde formed as a result of cleavage of the donor substrate from the thiazole ring of thiamine diphosphate to its aminopyrimidine ring through the tricycle formation stage, which is necessary for binding and splitting the second molecule of donor substrate [O.N. Solovjeva et al., The mechanism of a one-substrate transketolase reaction, Biosci. Rep. 40 (8) (2020) BSR20180246]. Here we show that under the action of the reducing agent a tricycle accumulates in a significant amount. Therefore, a significant decrease in the reaction rate of the one-substrate transketolase reaction compared to the two-substrate reaction is due to the stage of transferring the first glycolaldehyde molecule from the thiazole ring to the aminopyrimidine ring of thiamine diphosphate. Fragmentation of the four-carbon thiamine diphosphate derivatives showed that two glycolaldehyde molecules are bound to both coenzyme rings and the erythrulose molecule is bound to a thiazole ring. It was concluded that in the one-substrate reaction erythrulose is formed on the thiazole ring of thiamine diphosphate from two glycol aldehyde molecules linked to both thiamine diphosphate rings. The kinetic characteristics were determined for the two substrates, fructose 6-phosphate and glycolaldehyde.
Crystal structure of a xylulose 5-phosphate phosphoketolase. Insights into the substrate specificity for xylulose 5-phosphate
2019, Journal of Structural BiologyPhosphoketolases (PK) are TPP-dependent enzymes which play essential roles in carbohydrate metabolism of numerous bacteria. Depending on the substrate specificity PKs can be subdivided into xylulose 5-phosphate (X5P) specific PKs (XPKs) and PKs which accept both X5P and fructose 6-phosphate (F6P) (XFPKs). Despite their key metabolic importance, so far only the crystal structures of two XFPKs have been reported. There are no reported structures for any XPKs and for any complexes between PK and substrate. One of the major unknowns concerning PKs mechanism of action is related to the structural determinants of PKs substrate specificity for X5P or F6P. We report here the crystal structure of XPK from Lactococcus lactis (XPK-Ll) at 2.1 Å resolution. Using small angle X-ray scattering (SAXS) we proved that XPK-Ll is a dimer in solution. Towards better understanding of PKs substrate specificity, we performed flexible docking of TPP-X5P and TPP-F6P on crystal structures of XPK-Ll, two XFPKs and transketolase (TK). Calculated structure-based binding energies consistently support XPK-Ll preference for X5P. Analysis of structural models thus obtained show that substrates adopt moderately different conformation in PKs active sites following distinct networks of polar interactions. Based on the here reported structure of XPK-Ll we propose the most probable amino acid residues involved in the catalytic steps of reaction mechanism. Altogether our results suggest that PKs substrate preference for X5P or F6P is the outcome of a fine balance between specific binding network and dissimilar catalytic residues depending on the enzyme (XPK or XFPK) – substrate (X5P or F6P) couples.
Stages of the formation of nonequivalence of active centers of transketolase from baker's yeast
2019, Molecular CatalysisFor baker’s yeast transketolase (TK), cooperative binding of thiamine diphosphate (ThDP) and substrates in the transferase reaction is known. We show here that the differences in the properties of the active centers of TK are formed already upon the binding of Ca2+ in one of two initially identical subunits. When Ca2+ is bound in only one of the two active centers its affinity for the second decreases. The absence of a cation in the second active center decreases the affinity of ThDP to the first active center. Ca2+ binding increases the thermal stability of apo- and holoTK, i.e. changes the whole structure of the enzyme. Only in the presence of Ca2+, but not Mg2+, does the thermal stability of holoTK increase.
In the one-substrate reaction in the presence of Ca2+, two Km are measured for the binding of xylulose-5-phosphate and hydroxypyruvate. For both substrates, Vmax of the first active center of holoTK, when it binds the substrate alone, is higher than of semiholoTK. When the substrate begins to bind also in the second active center, Vmax of both active centers decreases, which is explained by the previously shown flip-flop mechanism.
Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii
2017, Biochimica et Biophysica Acta - General SubjectsIn photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg2 +.
TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography.
CrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg2 +. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg2 +-dependent and proceeds with a slow kinetics. The 3D–structure of CrTK without cofactors (CrTKapo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg2 + participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation.
These evidences indicate that Mg2 + is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg2 + is involved in the control of redox sensitivity of CrTK.
The importance of Mg2 + in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg2 + in chloroplasts.
Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase
2016, Enzyme and Microbial TechnologyImprovement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.