Thiamin Diphosphate Activation in 1-Deoxy-d-xylulose 5-Phosphate Synthase: Insights into the Mechanism and Underlying Intermolecular Interactions

Abstract

1-Deoxy-D-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4′-amino-pyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4′-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ~10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.

The Catalytic Mechanism of Human Transketolase

“…In physiological conditions, it was observed that hTK requires the presence of the thiamine diphosphate (ThDP) cofactor as co-catalytic agent. [1,2,[32][33][34] An example of a reaction catalyzed by hTK is reported in Scheme 1, which also represents the reaction studied in the present work. [1,13,[17][18][19][20] Since the product of this last process is a highly reactive species, ThDP-dependent enzymes are widely adopted in relevant processes for several chemoenzymatic synthesis involving the formation or the cleavage of CÀ S, CÀ N, CÀ C and CÀ O bonds, [21][22][23][24][25][26][27][28] as well as the sustainable and ecofriendly asymmetric carboligation of both aliphatic and aromatic compounds.…”

“…Upon binding, X5P engages in several hydrogen bonds with the active site sidechains, as evidenced in Figure 1. [1,18,33,39] In order to prevent large artificial movements of the amino acids in the quantum cluster model, the coordinate-locking scheme has been adopted in which amino acid residues are usually truncated at Cα atoms and these are fixed during geometry optimizations, being the overall structure close to the experimental one. Atom O4 is involved in hydrogen bonds with residues Asp424B, His37A and His109 A, while atom O3 is involved in a hydrogen bond with His258A.…”

“…[1,13,[17][18][19][20] Since the product of this last process is a highly reactive species, ThDP-dependent enzymes are widely adopted in relevant processes for several chemoenzymatic synthesis involving the formation or the cleavage of CÀ S, CÀ N, CÀ C and CÀ O bonds, [21][22][23][24][25][26][27][28] as well as the sustainable and ecofriendly asymmetric carboligation of both aliphatic and aromatic compounds. [1,32,33] In particular, the ultra-high-resolution crystal structures revealed a specific elongation of the CÀ C scissile bonds that link the cofactor to ThDP, with bond distance d > 1.60 Å. The reaction proceeds with the release of the first product and, in the presence of the second substrate (the aldose), the second product is formed (stage two).…”

“…The reaction proceeds with the release of the first product and, in the presence of the second substrate (the aldose), the second product is formed (stage two). [1,2,[32][33][34] An example of a reaction catalyzed by hTK is reported in Scheme 1, which also represents the reaction studied in the present work. It consists of the transfer of a dihydroxyethyl group from the ketose Dxylulose-5-phosphate (X5P) to the aldose D-erythrose-4phosphate (E4P), to yield D-fructose-6-phosphate (F6P) and Dglyceraldehyde-3-phosphate (G3P), as products.…”

“…[32] The sub-ångström resolution provided structural features and the existence of a substrate-ThDP covalent intermediate was confirmed, [32] corroborating previous experimental indications. [1,32,33] In particular, the ultra-high-resolution crystal structures revealed a specific elongation of the CÀ C scissile bonds that link the cofactor to ThDP, with bond distance d > 1.60 Å. [32] Stimulated by this interesting and unusual catalytic enzymatic mechanism, we have studied the reaction mechanism of hTK employing density functional theory (DFT), in order to establish the atomic details of the entire catalytic cycle.…”