Helicobacter pylori FabX contains a [4Fe-4S] cluster essential for unsaturated fatty acid synthesis

Abstract

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa.

The Molecular Basis of Catalysis by SDR Family Members Ketoacyl‐ACP Reductase FabG and Enoyl‐ACP Reductase FabI in Type‐II Fatty Acid Biosynthesis

“…Superposition of the FabG and FabI ternary complexes demonstrated clear differences in the ACP binding mode of each enzyme, although ACP interacts with them using a similar α2 helix, which is consistent with previous observations on other enzymes, [23,29,30] leading to the alternative insertion of ACP Ppant-acyl arm into the grooves through a Y-shaped tunnel (Figures 4c and 5a). ACP bound to two adjacent FabG monomers through interactions between the ACP α2 helix and the FabG α7 helices.…”

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target

“…Across the four mutants, three classes of resistance-associated mutations arose as follows: in the ORF for fabK , its upstream regulatory region, and/or the upstream region of C4N14_08920 (which encodes a putative nitronate monooxygenase that is homologous to the Helicobacter pylori FabX, a bifunctional dehydrogenase/isomerase, which synthesizes unsaturated fatty acids). 49 C4N14_08920 aligns with the H. pylori FabX, sharing 50.8% identity and 69.6% similarity. C4N14_08920 also possesses the key cysteine residues for the formation of the [4Fe–4S] clusters and residues that are important for FMN binding ( Figure S4 ).…”

“…All genomes were deposited in NCBI under accession number PRJNA1028625. Across the four mutants, three classes of resistance-associated mutations arose as follows: in the ORF for fabK , its upstream regulatory region, and/or the upstream region of C4N14_08920 (which encodes a putative nitronate monooxygenase that is homologous to the Helicobacter pylori FabX, a bifunctional dehydrogenase/isomerase, which synthesizes unsaturated fatty acids) . C4N14_08920 aligns with the H.…”

Barcoded overexpression screens in gut Bacteroidales identify genes with new roles in carbon utilization and stress resistance

“…67 Finally, a beneficial nitronate monooxygenase flavoprotein was identified to be FabX as it is similar to HP0773 (∼40% identity) from Helicobacter pylori . 68, 69 FabX is essential for unsaturated fatty acyl biosynthesis and requires molecular oxygen as the electron acceptor in vitro . 68, 69 However, a homolog in Neisseria gonorrhoeae (NGO1024, ∼36% identity) is essential for anaerobic unsaturated fatty acyl biosynthesis and relies on an unknown electron acceptor.…”

“…68, 69 FabX is essential for unsaturated fatty acyl biosynthesis and requires molecular oxygen as the electron acceptor in vitro . 68, 69 However, a homolog in Neisseria gonorrhoeae (NGO1024, ∼36% identity) is essential for anaerobic unsaturated fatty acyl biosynthesis and relies on an unknown electron acceptor. 70 We cloned out BCAT, VOR, and FabX and confirmed they are beneficial in individual growth assays (Fig.…”