Figure 1.
DHNA-CoA synthase (MenB) in the menaquinone biosynthesis.
(A) The bacterial biosynthetic pathway of menaquinone (vitamin K2). SEPHCHC: (1R, 2S, 5S, 6S)-2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylate; OSB: o-succinyl-1-benzoate; DHNA: 1, 4-dihydroxy-2-naphthanoate; CoA: coenzyme A. (B) Intermediates in the proposed catalytic mechanism for DHNA-CoA synthase.
Table 1.
Data collection and refinement statistics.a
Figure 2.
Overall structure of the ecMenB: HNA-CoA complex.
(A) The hexameric assembly of ecMenB:HNA-COA complex viewed along the 3-fold axis of the trimers. The protein is colored by chain and HNA-CoA is shown in sphere. (B) Side view of the hexamer in (A) along the trimer-trimer interface. (C) Superposition of a typical subunit of ecMenB: HNA-CoA (in salmon and red with the ligand in spheres) and a subunit of apo-ecMenB (PDB code: 4ELX, in cyan). The ordered A-loop (residues 88–105) and the re-oriented C-helix (residues 260–272) are highlighted in red. (D) Partial conformational changes in a ligand-binding subunit (chain B in green and marine) and its opposing unliganded subunit (chain D in yellow and red) across the trimer-trimer interface. Chain B exhibits an ordered A-loop (in marine) and an unchanged C-helix (in marine), whereas chain D contains a disordered A-loop (the two ends are indicated by arrows) and a reoriented C-helix (in red). The HNA-CoA ligand in chain B is shown in sticks.
Figure 3.
Interactions between the acyl moiety of the ligands and active site residues in ecMenB (A) and scMenB (B) complexes.
Side chains of the conserved active site residues are shown in sticks with carbon atom colored yellow. Water molecules are shown in red spheres and chloride anion is shown in green sphere. Dashed lines indicate potential hydrogen bonds with a distance less than 3.5 Å. In (A), the protein structure of ecMenB: HNA-CoA is represented in gray cartoon and HNA-CoA is represented as sticks with C, O, N, S and P atoms colored light blue, red, blue, brown and orange, respectively. The substrate analog OSB-NCoA and the side chains of Tyr-258 and Tyr-97 from the ecMenB: OSB-NCoA structure (PDB code: 3T88) are superimposed onto the ecMenB:HNA-CoA structure and shown with all carbon atoms colored in pink. In (B), the protein structure of scMenB: SA-CoA is represented in gray cartoon and the ligands SA-CoA and bicarbonate are represented as sticks with C, O, N, S and P atoms colored pink, red, blue, brown and orange, respectively. HNA-CoA from the scMenB:HNA-CoA structure is superimposed onto the scMenB:SA-CoA structure and shown with all carbon atoms colored in light blue.
Figure 4.
The interactions between the ordered A-loop and the reoriented C-helix in ecMenB:HNA-CoA (A) and scMenB:HNA-CoA (B).
Residues located in the loop-helix interface are displayed in sticks with carbon atoms colored yellow for the A-loop and green for the C-helix. Dashed lines represent distances less than 3.5 Å.
Figure 5.
New interactions between the coenzyme A thioesters and the ligand-induced loop-helix assembly in the ecMenB:HNA-CoA (A) and scMenB:SA-CoA (B) complexes.
The ordered A-loop is colored green and the C-helix is colored yellow. The loop connecting the second β-sheet and the first α-helix at the N-terminus, called loop 2, is colored blue. The ligands are represented as sticks with C, O, N, S and P atoms colored light blue, red, blue, brown and orange, respectively. Amino acid residues involved in interaction with the ligands are in stick representation with carbon atoms in the same color as the substructures. Dashed lines represent distances less than 3.5 Å.
Table 2.
Kinetic constants of the ecMenB mutants.
Figure 6.
Conservation of the MenB residues involved in the induced loop-helix and protein-ligand interactions.
These residues are distributed on the A-loop, C-helix, and Loop 2: the purple residues are conserved among >95% of MenB orthologues and directly involved in the ligand induced interactions; the green residue is a strictly conserved active-site tyrosine; the red residues are conditionally conserved among 113 of the 140 MenB sequences identified in Ref. 18 to form a hydrogen bond between the A-loop and the ribose ring of the ligand as observed for ecMenB (Figure 5A); and the blue residues are conditionally conserved among 23 of the 140 MenB sequences identified in Ref. 18 to form an lp−π interaction as observed for scMenB (Figure 5B). The presented sequences are aligned using Clustal W [55]. The secondary structures above the sequences are from the ecMenB:HNA-CoA structure, which are represented in arrows for β-pleated sheets, in rectangles for α-helices, and in lines for random coils.