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

Jiashen Zhou,Liang Zhang,Pan Zhang,Lu Yu,Hongkai Bi,Yuanyuan Duan ,John E Cronan,Siqi Shen,Lu Zhou,Jing Jiang,Xudong Hang,Liping Zeng,Jingyan Hu,Hou‐Wen Lin ,Lin Zhang,Wenyan Song,Yinan Zhang,Xiaoxue Ruan,Changlin Tian,Hongzhuan Chen ,Jia Jia

doi:10.1038/s41467-021-27148-0

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.

Highlights

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria
To provide a means to allow survival of H. pylori strains lacking the fabX gene, we introduced a synthetic copy of the E. coli fabA gene driven by a H. pylori promoter
Expression of E. coli FabA allowed the H. pylori fabX gene to be readily deleted (Fig. S1) and the replacement of FabX by FabA did not affect the growth of H. pylori (Fig. S2)

Summary

Introduction

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. 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 His[182] 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 anaerobic pathways generally proceed together with or after the dehydration step of the elongation cycle by utilizing either a dehydratase/isomerase bifunctional enzyme such as Escherichia coli FabA (in most Gram-negative bacteria)[7,12], FabN a Synthesis initiation ACP apo-ACP AcpS. NADH trans-2-C10:1-ACP cis-3-C10:1-ACP (in Enterococcus faecalis and Tetracococcus salidis)[13], Aerococcus viridans FabQ14, or a mono-functional isomerase, such as Streptococcus pneumoniae FabM15 to generate the cis-double bond in all cases followed by acyl chain elongation[10,11,16,17]

Methods

Results

Conclusion