| 2006 |
PfFabG (3-oxoacyl-ACP reductase) was crystallized at 1.5 Å resolution, revealing a homotetrameric structure with a catalytic triad of serine, tyrosine, and lysine aligned in an active conformation; a sulphate ion occupies the cofactor-binding site; and a cluster of positively charged residues at the active-site entrance is consistent with ACP substrate recognition. Kinetic and product inhibition studies indicated a steady-state ordered mechanism, with NADPH binding to the free enzyme first. Activity with NADH was <3% of that with NADPH. Hexachlorophene inhibited FabG with IC50 2.05 µM via non-linear competitive inhibition with respect to NADPH. |
X-ray crystallography (1.5 Å), fluorescence enhancement binding assays, kinetic analysis (steady-state ordered mechanism), product inhibition, in vitro enzymatic assay, inhibitor titration |
The Biochemical journal |
High |
16225460
|
| 2003 |
Recombinant P. falciparum FabG expressed in E. coli is soluble and enzymatically active, catalyzing reduction of acetoacetyl-CoA with Km 75 µM, Vmax 0.0054 µmol/min/ml, and kcat 0.014 s−1. NADPH exhibits negative cooperativity in binding to FabG. Homology modeling using Brassica napus FabG as template provided a structural rationale for NADPH cofactor specificity. |
Recombinant protein expression in E. coli, in vitro enzymatic assay with acetoacetyl-CoA, kinetic characterization, homology modeling |
Biochemical and biophysical research communications |
Medium |
12646215
|
| 2006 |
Binding of NADPH to PfFabG increases the binding affinity of substrate acetoacetyl-CoA by 16-fold and increases affinity for acyl carrier protein (ACP) approximately 3-fold, demonstrating ordered cofactor-mediated activation. PfFabG exhibits negative homotropic cooperativity for NADPH, which is enhanced in the presence of ACP. ACP increases NADPH accessibility to PfFabG. In the presence of NADP+, the reverse reaction is also observed (Ka = 23.17 µM−1). |
Intrinsic fluorescence spectroscopy (Trp334 nm), NADPH fluorescence (450 nm), acrylamide quenching assays, binding affinity measurements |
The FEBS journal |
Medium |
16934037
|
| 2008 |
Arg187 and Arg230 of PfFabG are critical residues for interaction with P. falciparum ACP (PfACP). Mutagenesis of Arg187, Arg190, and Arg230 reduced affinities of PfFabG mutants for acetoacetyl-ACP (the physiological substrate) compared to wild-type, while activity with the substrate analog acetoacetyl-CoA was retained. Surface plasmon resonance and intrinsic fluorescence confirmed direct PfFabG–PfACP binding, identifying a positively charged/hydrophobic patch adjacent to the active site as the ACP interaction site. |
Site-directed mutagenesis, ACP-dependent and ACP-independent enzymatic assays, intrinsic fluorescence binding assays, surface plasmon resonance |
The FEBS journal |
High |
18721141
|
| 2007 |
The elongation module of P. falciparum FAS-II was reconstituted in vitro using all four enzymes—FabB/F, FabG, FabZ, and FabI—and the reaction products were analyzed by MALDI-TOF MS. The distribution of acyl products from the reconstituted system matched the in vivo pattern, confirming FabG participates sequentially in iterative fatty acid chain elongation. Inhibition of FabG by (-)-catechin gallate led to accumulation of the upstream intermediate, detectable by MS. |
In vitro reconstitution of FAS-II elongation module, MALDI-TOF MS product analysis, inhibitor perturbation assay |
Antimicrobial agents and chemotherapy |
High |
17485508
|
| 2007 |
PfFabG is a homotetramer (Mr ~120,000) belonging to the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Guanidinium chloride-induced denaturation follows a simple A4 ↔ 4A two-state unfolding model. Conformational stability (ΔGs) is 22.68 kcal/mol with ΔCp of 5.83 kcal/(mol·K), attributed to the large tetrameric size and buried hydrophobic core. |
Intrinsic fluorescence spectroscopy, circular dichroism, denaturant-induced unfolding, enzymatic activity measurements, thermodynamic analysis |
IUBMB life |
Medium |
17654120
|
| 2008 |
Urea-induced unfolding of PfFabG is a multi-step process, whereas GdmCl-induced unfolding is two-state. In the presence of NADPH during urea unfolding, an activity-enhanced intermediate state is populated at ~1 M urea (90% activity recovered upon dilution), while NADPH provides no stabilization during GdmCl unfolding. NADPH binding leads to compaction of PfFabG structure as evidenced by decreased tryptophan accessibility to acrylamide quenching. |
Intrinsic fluorescence, circular dichroism, enzymatic activity measurement, glutaraldehyde cross-linking, size exclusion chromatography, acrylamide quenching |
Proteins |
Medium |
17879351
|
| 2006 |
Flavonoids inhibit PfFabG in a noncompetitive manner (luteolin and (-)-catechin gallate used as model compounds in kinetic analysis), establishing the mode of inhibition at the enzyme level. C-3 gallic acid esters of catechins are the most potent inhibitors (IC50 0.2–1.1 µM). |
In vitro enzyme inhibition assay, kinetic analysis (mode of inhibition determination), IC50 determination |
Journal of medicinal chemistry |
Medium |
16722653
|
| 2005 |
PfFabG overexpressed in E. coli as inclusion bodies could be correctly refolded only in the presence of the cofactor NADPH and 300 mM NaCl, demonstrating that NADPH is required for proper folding of the recombinant enzyme. The refolded protein was enzymatically active with acetoacetyl-CoA. |
Recombinant protein expression, inclusion body refolding, enzymatic activity assay |
Protein expression and purification |
Low |
15939298
|