Article

Crystallization and preliminary X-ray diffraction analysis of the high molecular weight ketoacyl reductase FabG4 complexed with NADH.

Department of Biotechnology, Indian Institute of Technology, Kharagpur, Kharagpur 721 302, India.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.57). 07/2012; 68(Pt 7):786-9. DOI: 10.1107/S1744309112020301
Source: PubMed

ABSTRACT FabG4 from Mycobacterium tuberculosis belongs to the high molecular weight ketoacyl reductases (HMwFabGs). The enzyme requires NADH for β-ketoacyl reductase activity. The protein was overexpressed, purified to homogeneity and crystallized as a FabG4-NADH complex. A mountable FabG4:NADH complex crystal diffracted to 2.59 Å resolution and belonged to space group P1, with unit-cell parameters a = 63.07, b = 71.03, c = 92.92 Å, α = 105.02, β = 97.06, γ = 93.66°. The Matthews coefficient suggested the presence of four monomers in the unit cell. In addition, a self-rotation function revealed the presence of two twofold NCS axes and one fourfold NCS axis. At χ = 180° the highest peak corresponds to the twofold NCS between two monomers, whereas the second peak corresponds to the twofold NCS between two dimers.

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    ABSTRACT: FabGs or β-ketoacyl reductases are involved in fatty acid synthesis. The reaction entails NADPH/NADH mediated conversion of β-ketoacyl-ACP to β-hydroxyacyl-ACP. High molecular weight FabGs (HMwFabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG of Mycobacterium tuberculosis, comprises of two distinct domains Nterminal "flavodoxin type" domain and C-terminal ketoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-ketoacyl-CoA to β-hydroxyacyl-CoA. Here the crystal structures of FabG4:NADH binary complex and FabG4:NAD+:HexanoylCoA ternary complex have been determined to understand its substrate specificity and catalytic mechanism. 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 coenzyme A. 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). The ternary complex reveals that both loop-I and loop-II interacts with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural evidences and ACP inhibition studies indicates that FabG4 can accepts both CoA and ACP based fattyacyl substrates. We have also shown that in 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.
    Biochemical Journal 11/2012; · 4.78 Impact Factor

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