Structural Insights into the Mechanism and Inhibition of the β-Hydroxydecanoyl-Acyl Carrier Protein Dehydratase from Pseudomonas aeruginosa

Abstract

Fatty acid biosynthesis is an essential component of metabolism in both eukaryotes and prokaryotes. The fatty acid biosynthetic pathway of Gram-negative bacteria is an established therapeutic target. Two homologous enzymes FabA and FabZ catalyze a key step in fatty acid biosynthesis; both dehydrate hydroxyacyl fatty acids that are coupled via a phosphopantetheine to an acyl carrier protein (ACP). The resulting trans-2-enoyl-ACP is further polymerized in a processive manner. FabA, however, carries out a second reaction involving isomerization of trans-2-enoyl fatty acid to cis-3-enoyl fatty acid. We have solved the structure of Pseudomonas aeruginosa FabA with a substrate allowing detailed molecular insight into the interactions of the active site. This has allowed a detailed examination of the factors governing the second catalytic step. We have also determined the structure of FabA in complex with small molecules (so-called fragments). These small molecules occupy distinct regions of the active site and form the basis for a rational inhibitor design program.

FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

“…The purified protein had an apparent monomeric molecular mass of ~18 kDa based on SDS-gel electrophoresis. The size exclusion chromatographic elution profile of FabQ (Figure S1 available online) indicated that the FabQ is a hexamer in solution form as are the FabZ proteins of E. coli and P. aeruginosa (Kimber et al, 2004), whereas the E. coli and P. aeruginosa FabA proteins are dimeric both in solution (Kass et al, 1967; Kimber et al, 2004) and in crystals (Leesong et al, 1996; Moynié et al, 2013). …”

“…Recently an unidentified anaerobic UFA synthesis mechanism involving a gene called ufaA was reported in Neiserria gonorrhoeae (Isabella and Clark, 2011), which may require a partner (perhaps a specific electron transport chain). Anaerobic synthesis of UFAs is unique to bacteria and thus the key enzymes of this pathway, particularly FabA, have been targeted for development of new antibacterial agents (Ishikawa et al, 2012; Moynié et al, 2013). …”

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

“…[136] The NAC molecule hydrogen-bonds with loop C as well as with a water molecule bridging the other subunit. The NAC moiety binds closely to catalytic residues Asp84 and His70 (the same residues found in Ec FabA).…”

“…Pa FabA and Ec FabA structures shows this loop contributes extensively to the alkyl-binding tunnel, whereas the α3-β3 loop is the major contributor to the alkyl-binding tunnel in FabZ. [136] …”

“…Two fragments were found to block the pantetheine-binding tunnel and partly mimic the flavonoids and synthetic inhibitors reported for Hp FabZ. [136] …”