The thiamin diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d-glyceraldehyde 3-phosphate (d-GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d-GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor-acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism.

The mechanism of a one-substrate transketolase reaction. Part II

Endocytosis, signal transduction and proteolytic cleaving of human holotransferrin in Entamoeba histolytica.

Transketolase catalyzes the interconversion of keto and aldo sugars. Its coenzyme is thiamine diphosphate. The binding of keto sugar with thiamine diphosphate is possible only after C2 deprotonation of its thiazole ring. It is believed that deprotonation occurs due to the direct transfer of a proton to the amino group of its aminopyrimidine ring. Using mass spectrometry, it is shown that a water molecule is directly involved in the deprotonation process. After the binding of thiamine diphosphate with transketolase and its subsequent cleavage, a thiamine diphosphate molecule is formed with a mass increased by one oxygen molecule. After fragmentation, a thiamine diphosphate molecule is formed with a mass reduced by one and two hydrogen atoms, that is, HO and H2O are split off. Based on these data, it is assumed that after the formation of holotransketolase, water is covalently bound to thiamine diphosphate, and carbanion is formed as a result of its elimination. This may be a common mechanism for other thiamine enzymes. The participation of a water molecule in the catalysis of the one-substrate transketolase reaction and a possible reason for the effect of the acceptor substrate on the affinity of the donor substrate for active sites are also shown.

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New Role of Water in Transketolase Catalysis

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

Dmitrii S Shcherbinin

Papers

The mechanism of a one-substrate transketolase reaction.