A Thiamine-Dependent Enzyme Utilizes an Active Tetrahedral Intermediate in Vitamin K Biosynthesis

Abstract

Enamine is a well-known reactive intermediate mediating essential thiamine-dependent catalysis in central metabolic pathways. However, this intermediate is not found in the thiamine-dependent catalysis of the vitamin K biosynthetic enzyme MenD. Instead, an active tetrahedral post-decarboxylation intermediate is stably formed in the enzyme and was structurally determined at 1.34 Å resolution in crystal. This intermediate takes a unique conformation that allows only one proton between its tetrahedral reaction center and the exo-ring nitrogen atom of the aminopyrimidine moiety in the cofactor with a short distance of 3.0 Å. It is readily convertible to the final product of the enzymic reaction with a solvent-exchangeable proton at its reaction center. These results show that the thiamine-dependent enzyme utilizes a tetrahedral intermediate in a mechanism distinct from the enamine catalytic chemistry.

Single‐Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate‐Dependent Catalysis in Vitamin K Biosynthesis

“…As reported previously, the distinct tetrahedral Breslow intermediate in MenD catalysis shows a sharp positive band at λ =302 nm and a broad negative band from λ =305 to 365 nm with double minima at λ =320 and 334 nm by circular dichroism (CD) spectroscopy; this is different from a broad positive band in the ranges of λ =290 to 295 and λ >300 nm that are characteristic of the enamine intermediate in transketolase . These characteristic CD bands are due to the charge‐transfer excitation between the pyrimidine ring and the thiazolium ring of the cofactor, and both were used in our pre‐steady‐state kinetic analysis of the various intermediates in MenD catalysis.…”

“…This requires that the intermediate is stable enough to withstand exposure to an aqueous environment without being damaged in wait of the incoming electrophilic substrate. Our previous tests demonstrated that this intermediate had the required stability to maintain its characteristic CD spectrum intact for up to minutes without any significant loss in its activity . This high stability strongly supports that the reactive intermediate predominantly takes the neutral tautomeric form with a protonated C 2α center in its equilibrium with the much more unstable carbanion tautomer (Scheme ).…”

“…However, owing to the low biological availability of isochorismate, this rate‐limiting step may be changed and the reactive intermediate E‐Int is likely accumulated in the catalysis. Together with identification of the distinct ThDP‐bound tetrahedral Breslow intermediate, these findings lay the foundation for a new ThDP‐dependent catalytic mode distinct from the canonical enamine chemistry which may be explored for the development of new antibiotics against the essential ThDP‐dependent enzyme in menaquinone biosynthesis.…”

“…Protein preparation and activity assay : Escherichia coli MenD was overexpressed and purified to homogeneity as an untagged protein by using a HiPrep DEAE FF 16/10 column (GE Healthcare) and a HiPrep Sephacryl S‐200 column (GE Healthcare) as described previously . For activity assay, the isochorismate substrate was chemoenzymatically prepared chorismic acid by using EntC and was purified by high‐performance reverse‐phase HPLC by using a semipreparative C 18 column .…”

“…After centrifugation at 20 000 rpm in a FA‐45‐18‐11 rotor for 5 min at 4 °C in an Eppendorf Centrifuge 5418R to remove the protein precipitates, the mixture was filtered through a Millipore Ultrafiltration unit with a molecular weight cutoff of 3000, and the filtrate was separated and purified before MALDI‐TOF analysis by using an Xterra semipreparative C 18 reversed‐phase column (10 μm, 7.8×300 mm) and isocratic elution with water containing 1 % formic acid at a flow rate of 2 mL min −1 . As a positive control, the same reaction was quenched at 2 min, a time long enough to fully form the tetrahedral intermediate (E‐Int), and the mixture was processed and analyzed for comparison. Another control reaction was also run without the addition of MenD and was quenched at 50 ms for parallel analysis.…”