Mechanistic and structural insights into the itaconate-producing trans-aconitate decarboxylase Tad1

Abstract

Itaconic acid belongs to the high-value precursors for the production of biomass-based industrial compounds. It originates from the tricarboxylic acid cycle, and depending on the organism, it is produced by different biosynthetic routes. The basidiomycete fungus Ustilago maydis synthesizes itaconic acid via isomerization of cis-aconitic acid to trans-aconitic acid, and subsequent decarboxylation catalyzed by the trans-aconitate decarboxylase Tad1, which belongs to the aspartase/fumarase superfamily. Since no other decarboxylase has been identified within this protein superfamily, Tad1 constitutes a novel type of decarboxylase. Here, we present high-resolution crystal structures of Tad1, which, together with mutational analysis and nuclear magnetic resonance spectroscopy measurements, provide insight into the molecular mechanism of Tad1-dependent decarboxylation. Specifically, our study shows that decarboxylation is favored in acidic conditions, requires protonation as well as migration of a double bond, and coincides with structural rearrangements in the catalytic center. In summary, our study elucidates the molecular mechanism underlying a novel type of enzymatic decarboxylation and provides a starting point for protein engineering aimed at optimizing the efficient production of itaconic acid.

Fig. 1.

Purification and functional characterization of Tad1. A) Biosynthesis of IA (2) in U. maydis is achieved through initial isomerization of cis-aconitate to tA (1) catalyzed by Adi1 and subsequent decarboxylation of trans-aconitate by Tad1. B) Phylogenetic analysis of Tad1 shows its relationship to other members of the aspartase/fumarase superfamily, including CreD (bold), a highly homologous enzyme identified through a distance matrix alignment (Dali) search. NCBI accession numbers or PDB codes are shown. CMLE, 3-carboxy-cis,cis-muconate lactonizing enzyme; ASL, argininosuccinate lyase; ADSL, adenylosuccinate lyase. C) HPLC results showing Tad1 activity across a pH range of 5.5 to 9.5, presented from top to bottom as (i) to (v). D) The results of an LC–MS experiment performed on the products of Tad1 reactions in D₂O/H₂O, with 90% D₂O (top) and H₂O (bottom). E) ¹H-NMR spectra of IA produced by Tad1 in 90% D₂O (top) compared with the standard (bottom).

Fig. 2.

Overview of the Tad1 purification and structure. A) SEC-MALS analysis of Tad1 showing elution around 11.5 mL, representing an approximate MW of 220 kDa. B) Domain structure of Tad1, featuring domains D1, D2, and D3 from N- to C-terminus. The SS loop (S321 and S322) is located in the D2 region. C) Crystal structure of Tad1, depicted as a homotetramer with chains A (yellow), B (green), C (cyan), and D (magenta). D) Domain arrangement of D1, D2, and D3, including the SS loop, within a single chain of Tad1.

Fig. 3.

Identification and characterization of the Tad1 catalytic center. A) Multiple sequence alignment of central catalytic residues (E359 to P373) in Tad1 (decarboxylase), CreD (nitrosuccinate lyase), and CMLEs (PDB IDs 2FEL, 1RE5, and 1Q5N). The arginine residue, which could be a conserved catalytic residue, is highlighted with an arrow. B) Conversion rates of tA to IA for indicated Tad1 mutants are shown. The error bars represent the SD from three independent experiments. C–E) 2F₀−F_c electron density maps showing critical binding residues in Tad1. C) The interaction of tA with holo_Tad1_R360A. D) Glycerol binding in WT Tad1 (Tad1_WT). E) The structure of Tad1_S320A mutant in the presence of 20 mM tA. F) Comparative electron density map of tA (from C) and glycerol (from D). G) Sequence logo analysis of the SS loop region (G319 to N329) in Tad1 and the aspartase/fumarase family.

Fig. 4.

Proposed model of Tad1-catalyzed decarboxylation. A) Conformational changes in the catalytic center of Tad1. Structural comparison of apo-Tad1 (green) and holo-Tad1 (gray) in the presence of glycerol uncovers the dynamic adjustment of the active site upon substrate binding. B) Proposed coordination of tA within the Tad1 active center, modeled using Autodock Vina. C) A model for protonation and decarboxylation of tA.