Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

Abstract

β-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop β4-α4 and loop β5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants.

Importance: This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae.

FIG 1

Reduction of a β-ketoacyl group bound to a carrier protein to (3R)-3-hydroxyacyl by FabG, using NADPH as the reducing agent. ACP, acyl carrier protein.

FIG 2

Binding of NADPH measured by fluorescence assay. (A) Blueshift of NADPH fluorescence upon binding to vcFabG. Binding of NADPH to the vcFabG proteins exhibited a blueshift for the maximum emission wavelength from 460 nm to 445 nm, except for the case of the G92D mutant, for which no significant binding was detected. A representative profile of the fluorescence spectrum of 50 μM NADPH either free in solution or bound to vcFabG is shown. (B) Changes of fluorescence intensity measured at an emission wavelength of 445 nm for wild-type vcFabG and its mutants. The curves show the fits to the Hill equation (equation 1) for measurements for the wild-type protein and the G92A mutant protein. The changes in fluorescence intensity for the other mutants could not be fit due to the poor signal strengths.

FIG 3

Spectrophotometric kinetic analysis of vcFabG. (A) Activity of vcFabG in 1 mM AcAcCoA and different concentrations of NADPH. (B) Activity of vcFabG in 0.15 mM NADPH and different concentrations of AcAcCoA. (C) Kinetic activity of vcFabG mutants relative to the activity of the wild-type enzyme (defined as 100%). Each of the data points shown here is the mean from three separate experiments, and the error bars are standard deviations.

FIG 4

Analytical size-exclusion chromatography of vcFabG. The concentration of NaCl used in the experiments was 150 mM unless otherwise noted. (A) The NaCl concentration affects the oligomeric state of vcFabG. (B) NADP⁺ promotes the formation of the vcFabG tetramer. (C and D) Increasing the concentration of vcFabG promotes the formation of the tetramer. The fraction of the dimer in panel D was calculated using equation 4. It must be noted that calculation of the dimerization ratio was based on the hydrodynamic radius of the protein derived from the elution volume in the analytical gel filtration experiment and that the dimerization ratio is a qualitative rather than a quantitative measure. mAu, milli-absorbance units.

FIG 5

Binding of NADP(H) to vcFabG protein. (A) The cofactor binding pocket is highly conserved. The conservation scores were calculated by use of the ConSurf server (49) using the amino acid sequence of vcFabG and the structure of apo-vcFabG (PDB accession number 3RRO) as the search model. The BLAST program was used to search the NCBI GenBank nonredundant protein database. The maximum and minimum percentages of global identity between any two pairs of sequences were 92% and 52%, respectively. (B) NADP⁺ binds to vcFabG. The competent catalytic triad is formed by the organized side chains of S142, Y155, and K159. Water molecules and the side chains or main chains of amino acid residues which do not form a direct interaction with the cofactor were omitted for clarity. (C) Binding of NADPH in one monomer of the vcFabG(Y155F)/NADPH complex.

FIG 6

Comparison of loops β4-α4 and β5-α5 in the active and inactive conformations of vcFabG and ecFabG. In all the structures complexed with NADP(H), the ligand is shown in ball-and-stick mode. The residues responsible for activation of the enzymes (G92, G141, Q152, and Y155 in vcFabG and G88, G137, Q148, and Y151 in ecFabG) are highlighted in magenta. (A) vcFabG cocrystallized with NADP+, active conformation (PDB accession number 3OP4); (B) apo-vcFabG, active conformation (PDB accession number 3RRO); (C) vcFabG (Y155F) soaked with NADPH, inactive conformation (PDB accession number 3TZC); (D) ecFabG cocrystallized with NADP+, active conformation (PDB accession number 1Q7B); (E) apo-ecFabG, inactive conformation (PDB accession number 1I01); (F) ecFabG(Y151F) soaked with NADPH, active conformation (PDB accession number 1Q7C).

FIG 7

Dimer of dimers configuration of vcFabG. (A) The tetramer of vcFabG in the active conformation (PDB accession number 3OP4) is shown with the surface in light gray and the ribbon representation of each monomer in a different color. The two dimer interfaces are highlighted with arrows. The residues corresponding to changes occurring in the buried area upon conformational transitions are shown as purple dots. (B) Changes in dimer interface B from the active to the inactive conformation. The disordered region in the inactive conformation (cyan) and the corresponding region in the active conformation (green) are indicated by red ovals.

FIG 8

Characterization of key hinge residues in conformational transitions in FabG. (A) The conformations of loops β4-α4 and β5-α5 diverge between the active and inactive states in FabGs. All of the FabG structures shown are from PDB. All enzymes in the active state (shown in gray with PDB accession numbers 1Q7B, 1UZN, 2C07, 2P68, 2UVD, 3FTP, 3LYL, 3OP4, 3RRO, 3OSU, and 4AFN) share the same open conformation in the cofactor binding site, while all the enzymes in the inactive state (shown in blue with PDB accession numbers 1I01, 1UZL, 2NTN, 3F9I, 3GRP, and 3TZC) have disordered β4-α4 and β5-α5 loops, which appear to perturb the cofactor binding site and occlude the NADP(H) binding site. The NADP⁺ molecule bound to vcFabG is shown in ball-and-stick mode. (B) The conformations of loops β4-α4 and β5-α5 diverge between the active and inactive states at the G92 and G141 sites in vcFabG (the structure with PDB accession number 3OP4 is in gray, and the structure with PDB accession number 3TZC is in blue).

FIG 9

Active conformations of vcFabG(G92) mutants. (A and B) Ordered loops β4-α4 and β5-α5 in the structures of the G92D (A) and G92A (B) mutants. The amino acid residues critical for the activation of the enzyme are highlighted in magenta. (C) In the crystal structure of the vcFabG(G92D) mutant, the side chains of catalytic residues Y155 and K159 are locked in an active conformation by interactions with the side chain of D92. The presence of an Asp residue at this site appears to occlude binding of NADPH.

FIG 10

vcFabG(G141A) mutant structures. (A) Two out of four subunits in the structure of apo-vcFabG(G141A) are superposed to show the different orientations of loops β4-α4 and β5-α5 (dark green and pale green, respectively). The inactive conformation of the corresponding loops in MabA (PDB accession number 2NTN) is shown in yellow for comparison. (B) Disordered loops β4-α4 and β5-α5 in the apo-vcFabG(G141A) mutant structure. (C) Loops β4-α4 and β5-α5 in the structure of vcFabG(G141A) in complex with NADPH. Loop β4-α4 is more ordered than loop β4-α4 in the apo-vcFabG(G141A) mutant structure and adopts an orientation similar to that in the active conformation, while loop β5-α5 is still disordered and blocks the nicotinamide moiety from binding to the correct position.