RESUMO
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.
Assuntos
Proteínas de Bactérias/ultraestrutura , Ácidos Graxos Insaturados/biossíntese , Helicobacter pylori/enzimologia , Proteínas Ferro-Enxofre/ultraestrutura , Oxigenases de Função Mista/ultraestrutura , Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/genética , Cristalografia por Raios X , Helicobacter pylori/genética , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , OxirreduçãoRESUMO
We report crystal structures of the 2.6-megadalton alpha6beta6 heterododecameric fatty acid synthase from Thermomyces lanuginosus at 3.1 angstrom resolution. The alpha and beta polypeptide chains form the six catalytic domains required for fatty acid synthesis and numerous expansion segments responsible for extensive intersubunit connections. Detailed views of all active sites provide insights into substrate specificities and catalytic mechanisms and reveal their unique characteristics, which are due to the integration into the multienzyme. The mode of acyl carrier protein attachment in the reaction chamber, together with the spatial distribution of active sites, suggests that iterative substrate shuttling is achieved by a relatively restricted circular motion of the carrier domain in the multifunctional enzyme.