Phosphomethylpyrimidine Synthase (ThiC): Trapping of Five Intermediates Provides Mechanistic Insights on a Complex Radical Cascade Reaction in Thiamin Biosynthesis

Abstract

Phosphomethylpyrimidine synthase (ThiC) catalyzes the conversion of AIR to the thiamin pyrimidine HMP-P. This reaction is the most complex enzyme-catalyzed radical cascade identified to date, and the detailed mechanism has remained elusive. In this paper, we describe the trapping of five new intermediates that provide snapshots of the ThiC reaction coordinate and enable the formulation of a revised mechanism for the ThiC-catalyzed reaction.

Figure 1

Outline of thiamin biosynthesis.

Figure 2

(A) The ThiC-catalyzed reaction with the fate of all atoms shown in color. (B) Active site architecture of AtThiC showing residues involved in AIR binding (PDB: 4S28). (C) Active site of AtThiC showing the noncanonical [Fe₄S₄] cluster (CX₂CX₄C), a novel mononuclear iron site involved in SAM binding and bound S-adenosyl homocysteine (SAH).

Figure 3

Preliminary mechanistic proposal for the ThiC-catalyzed conversion of AIR (1) to HMP-P (2).

Figure 4

(A) Extracted Ion Chromatogram for the new product P₁₇ in the ThiC-catalyzed reaction. The two peaks observed for P₁₇ are the E and Z isomers of PFBHA-oxime 30. (B) MS analysis of P₁₇. (C) Retrosynthetic analysis of 30. (D) Summary of labeling studies to determine the origin of the atoms of compound 30.

Figure 5

(A) Revised mechanistic proposal for the early steps of the ThiC-catalyzed reaction. (B) Mechanistic proposal for the formation of shunt metabolite 31. (C) Proposed scheme for the hydrolysis of 35 in the shunt pathway.

Figure 6

(A) HPLC analysis of the CcThiC (E413Q) catalyzed reaction after PFBHA treatment showing the formation of P₂₄. (B) LC-MS analysis of P₂₄. (C) Retrosynthetic analysis of compound 40. (D) Summary of the labeling studies used to characterize 40 (E + Z). (E) Mechanistic proposal for the formation of 40 (E + Z).

Figure 7

Strategy for trapping formyl imidazole intermediate 39 (Figure 5) as 50 and the product of the first β-scission reaction 14 (Figure 3) as 55/56.

Figure 8

Reactions leading to the formation of 57 (E + Z). (A) HPLC analysis of the CcThiC (C474S) catalyzed reaction after the addition of PFBHA and (B) LC-MS of P_25.9 (57). (C) The CcThiC (C474S) catalyzed reaction and subsequent derivatization.

Figure 9

Labeling experiment that demonstrated that the shift of the C3′-H to C2′ occurred after the formation of the radical precursor to 58.

Figure 10

Proposed mechanism for the formation of the thiamin pyridine methyl group and the shunt products 58, 67, and 68.

Figure 11

Current mechanistic proposal for the ThiC-catalyzed reaction based on the trapping of five reaction intermediates derived from 14 (54), 33 (41), 34 (31), 39 (49), and 60 (58).

Figure 12

ThiC and the THI5-catalyzed biosynthesis of HMP-P.

Figure 13

(A) Biosynthesis of bacimethrin 79 and its conversion to methoxythiamin pyrophosphate 81 using late-stage thiamin biosynthetic enzymes. (B) Tautomerization of the thiamin pyrimidine to form the base (82) used in the formation of the thiamin ylide (83). (C) Mechanism of transketolase inhibition by MeO-TPP (Path 1) and proposed demethylation of MeO-TPP to form the 2′-oxythiamin pyrophosphate 84 antivitamin (Path 2).