Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

Abstract

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal 'flavodoxintype' domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4-NADH binary complex and the FabG4-NAD+-hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase

“…The ternary structure of a B-type KR complexed with a substrate is still unavailable. However, the structures of a high-molecular weight FabG complexed with hexanoyl-CoA (PDB: 3V1U) (Dutta et al, 2013) and a substrate-bound acetoacetyl-CoA reductase (PDB: 4N5M) (Kim et al, 2014) reveal that an aspartate equivalent to the third residue of the LDD motif of a B-type KR helps orientate the pantetheine handle by forming a hydrogen bond with the C4′ carbonyl group (Fig. 4B).…”

Biochemical Characterization of Isoniazid-resistant Mycobacterium tuberculosis: Can the Analysis of Clonal Strains Reveal Novel Targetable Pathways?

“…FabG4 has affinity for both NADH and NADPH cofactors and also for both Coenzyme A (CoA) and ACP fatty acid derivatives. FabG1 in turn, has a higher affinity for NADPH and almost exclusively ACP derivatives [61,62]. In addition to this, reduced levels of ACP derivatives (due to the reduced levels of AcpM) as well as the reduced levels of the HadABC complex enables the catalytic role of HtdX in this alternative FAS II route.…”

An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine

“…It should be noted that there is no a priori reason (such as active sites requiring interface residues) for FabG to be a tetramer. Indeed, a dimeric ␤-ketoacyl-ACP reductase is encoded by Synechocystis (21), and the solution structure of Pseudomonas aeruginosa RhlG, a ␤-ketoacyl-ACP reductase essential for rhamnolipid synthesis, has been reported to be dimeric by both size exclusion chromatography and analytical ultracentrifugation, although at high protein concentrations, tetramers were observed (22).…”