Enzymatic reaction mechanism of cis-aconitate decarboxylase based on the crystal structure of IRG1 from Bacillus subtilis

Abstract

Itaconate, which is formed by decarboxylation of cis-aconitate—an intermediate metabolite in the tricarboxylic acid cycle—has been used as a building block in polymer synthesis and is an important chemical in several biomedical and industrial applications. Itaconate is an immunometabolite with antibacterial, antiviral, immunoregulatory, and tumor-promoting activities. Recent focus has been on the role of itaconate in the field of immunology, with immune-responsive gene 1 (IRG1) being identified as the cis-aconitate decarboxylase responsible for itaconate production. We solved the structure of IRG1 from Bacillus subtilis (bsIRG1) and showed that IRG1 adopts either a closed or an open conformation; bsIRG1 was in the open form. A1 and A2 loops around the active site are flexible and can control the formation of the open and closed forms of IRG1. An in silico docking simulation showed that only the open form of IRG1 can accommodate the substrate. The most energetically favorable position of cis-aconitate in the active site of bsIRG1 involved the localization of C2 and C5 of cis-aconitate into the H102 region and H151 region of bsIRG1, respectively. Based on the structural study of bsIRG1, compared with IDS epimerase, and in silico docking simulation, we proposed two tentative enzymatic reaction mechanisms of IRG1, a two-base model and a one-base model.

The crystal structure of mouse IRG1 suggests that cis-aconitate decarboxylase has an open and closed conformation

“…This hypothesis led us to conclude that our mouse IRG1 is in the closed form. Indeed, recently, we solved the structure of another IRG1 from yeast; this structure was in an open form with the A1 loop located far from the active site [ 31 ]. In this study, we also showed that the lid domain, which can be tilted, might be flexible and adjust the active site, which is localized between the lid domain and the helical domain.…”

Aconitic Acid Recovery from Renewable Feedstock and Review of Chemical and Biological Applications

“…Some fungi naturally convert aconitic acid to itaconic acid through decarboxylation. For example, Aspergillus terreus is used in fermentations to produce itaconic acid [ 43 , 44 , 45 , 71 ]. In A. terreus, CAA is first transported by a mitochondrial transporter, At_MttA, from the TCA cycle in the mitochondria to the cytosol, where CAA is decarboxylated to itaconic acid by the cis -aconitic acid decarboxylase Cad-A [ 43 , 46 , 72 ].…”

“…For example, Aspergillus terreus is used in fermentations to produce itaconic acid [ 43 , 44 , 45 , 71 ]. In A. terreus, CAA is first transported by a mitochondrial transporter, At_MttA, from the TCA cycle in the mitochondria to the cytosol, where CAA is decarboxylated to itaconic acid by the cis -aconitic acid decarboxylase Cad-A [ 43 , 46 , 72 ]. Subsequently, itaconic acid is transported out of the mycelia by a specific transporter, belonging to the major facilitator superfamily (MFS) type transporter (MfsA) [ 73 ].…”

“…The genes involved in itaconic acid production in A. terreus also include a transcription factor, and the four genes are termed the “itaconate gene cluster” [ 73 ]. However, in the corn smut basidiomycete, Ustilago maydis, the mitochondrial transporter Um_Mtt1, also transports CAA from the mitochondria to the cytoplasm, but CAA is first isomerized to TAA by aconitate-Δ-isomerase, Adi1, then decarboxylated by trans -aconitic acid decarboxylase, Tad1, to itaconic acid [ 43 , 46 , 47 , 74 ]. The “itaconate gene cluster” in U. maydis is induced upon nitrogen limitation [ 75 ].…”

Itaconate: an antimicrobial metabolite of macrophages

“…Bacillus subtilis). 136 Nevertheless, its biological roles remain largely unknown. In addition to an immunomodulatory function of itaconate in mammals, several lines of evidence are supporting an antibacterial role in macrophages, including: 1) the known antibacterial activity of itaconate; 2) the reported itaconate inhibition of Icl, an enzyme essential for the survival of many pathogens in mammals; 137 Answers to these questions will likely reveal several new drug targets, not only for antimicrobials but also for disorders of the immune system.…”