Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode

Abstract

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family.

Keywords: crystal structure; enzyme catalysis; enzyme mechanism; substrate specificity; vitamin K.

Figure 1.

Catalysis of a two-step reaction by OSB-CoA synthetase (MenE) in two distinct active conformations. PP_i, pyrophosphate; CoA–SH, coenzyme A.

Figure 2.

Overall structure of the bsMenE–OSB-NCoA–AMP ternary complex. A, two-domain organization of bsMenE. B, surface and schematic representation of the functional dimer. Chain C is colored according to the domains shown in A, and the ligands OSB-NCoA and AMP are represented in ball-and-stick mode. C, tertiary folding of a bsMenE subunit in the ternary structure. The structure is presented in the same orientation as chain C in B with blue α-helices and green β-sheets in the N-domain and with magenta α-helices and yellow β-sheets in the C-domain. D, OSB-NCoA and AMP in chain A. The CoA 3′-phosphate interacts with three residues (Ser-220′, Val-221′, and Ser-222′) from the neighboring unit cell. E, OSB-NCoA and AMP in chain C. D and E, ligands are represented in ball-and-stick with 2mF_o − DF_c density map contoured at 2.0 σ (blue mesh) and 1.0 σ (orange mesh). F, superimposed OSB-NCoA ligands from chain C and chain A. The different orientation of the 3′-phosphate is highlighted. G, two views of the gauche ethylene group in the succinyl moiety of OSB-NCoA. The presented structure is highlighted in a red circle in E.

Figure 3.

Comparison of the bsMenE-OSB-NCoA-AMP structure in a thioesterification conformation with the bsMenE-OSB-AMP structure in an adenylation conformation. A, 139.5° difference in orientation of the C-domain (blue) between the two structures. The large N-domains (yellow) are superimposable and drawn in exactly the same way in the two structures, and the inter-domain linker (379–392) is colored in green. The two mutated residues I454R and A456K are denoted as spheres. The U-shaped OSB-AMP (green surface) represents the adenylate tunnel in adenylation conformation (left) and is fragmented into AMP (green surface) and OSB-CoA (represented by OSB-NCoA, white surface) in the thioesterification conformation (right). B, two views of the bsMenE-OSB-NCoA-AMP ternary structure in surface representation. The left view shows one end of the interconnected ligand-binding tunnel where AMP (sticks) is bound, and the right view shows the other end of the tunnel where the ADP moiety of the OSB-NCoA (sticks) is exposed to the bulk solution. The protein surface is colored according to domains as shown in A.

Figure 4.

Active site of the bsMenE thioesterification conformation. A, stereo diagram of the OSB-CoA-binding pocket. The ligand OSB-NCoA is represented as balls and sticks with its carbon atoms colored in pink. The nucleotide AMP is shown as magenta lines. The carbon atoms of the active-site residues from N-domain (1–379), linker (380–392), and C-domain (393–486) are respectively colored pale-cyan, green, and pale-yellow, except the two mutated residues I454R and A456K are highlighted in red. The water molecules that directly interact with OSB-NCoA or active-site residues are shown as red dots, and the magnesium ion is shown as a green sphere. The yellow dashed lines denote hydrogen bonds with a distance shorter than 3.5 Å. The oxygen, nitrogen, and phosphorus atoms in the whole panel are colored red, blue, and orange, respectively. The water molecules O1–O13 correspond to HOH14, HOH35, HOH1472, HOH791, HOH412, HOH238, HOH1513, HOH1482, HOH1055, HOH1821, HOH101, HOH875, and HOH239 of the ternary structure (chain C), respectively. The underlined residues have been mutated for kinetic studies. B, alignment of sequence fragments contributing to binding of the OSB moiety and the pantetheinyl group of OSB-NCoA. Secondary structural elements are shown for the bsMenE complex containing OSB-NCoA and AMP in the alignment. Residues conserved in 100, 100 to 95, and 95 to 80% sequences are represented with blue triangles, marked with red boxes, and shown in yellow background, respectively.

Figure 5.

Two CoA-binding modes in different ACS. A, 3′-phospho-ADP moiety of CoA binding to the C-domain in bsMenE (PDB code 5X8F). B, 3′-phospho-ADP moiety of CoA binding to the N-domain in S. enterica acetyl-CoA synthetase (PDB code 1PG4). C, 3′-phospho-ADP moiety of CoA binding to the N-domain in human medium chain acyl-CoA synthetase (ACSM2A, PDB code 3EQ6). D, 3′-phospho-ADP moiety of CoA binding to the N-domain in N. tabacum 4-coumarate-CoA ligase (PDB code 5BSR). The shown structures are from the same areas of the crystal structures of the ligand-bound proteins that have been aligned according to their N-domains. A–D, proteins are represented in schematic with a light-blue N-domain and a pale-yellow C-domain (left) and in electrostatic potential surface (right), and the CoA ligand is shown in balls-and-sticks, and the Mg²⁺ ion is presented as a green sphere. E, sequence alignment of the peptide fragments involved in binding of the 3′-phospho-ADP moiety. Structurally determined members with a CoA derivative are labeled with the corresponding PDB codes shown in parentheses (red). The red arrows point to the three conserved residues or their equivalents in the sequence alignment, which are shown or labeled in A–D.

Figure 6.

Modeling of the OSB-AMP intermediate into the thioesterification conformation. A, superimposed bsMenE-OSB-AMP and the IRAK–OSB-NCoA–AMP structure. The structures are colored in gray for the former (chain A, PDB code 5GTD) and pale-green for the latter (chain C, PDB code 5X8F). Ligands OSB-AMP, OSB-NCoA, and AMP are represented as balls-and-sticks. P-loop and the helical segment (Ala-238–Val-239–Gln-240–Thr-241) are different in the two structures and are shown in the schematic. The His-196 side chain takes a different orientation in the two structures, and both conformations are shown in sticks, and its hydrogen bonds with Glu-392 (in sticks) are denoted by yellow dashed lines. B, amplified view of OSB-NCoA and the modeled OSB-AMP. The area shown corresponds to the rectangular region circled by red dashes (A) with a slightly adjusted perspective.