Common basis for the mechanism of metallo and non-metallo KDO8P synthases

Abstract

The three-dimensional structures of metal and non-metal enzymes that catalyze the same reaction are often quite different, a clear indication of convergent evolution. However, there are interesting cases in which the same scaffold supports both a metal and a non-metal catalyzed reaction. One of these is 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes the synthesis of KDO8P and inorganic phosphate (P(i)) from phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and water. This reaction is one of the key steps in the biosynthesis of bacterial endotoxins. The evolutionary tree of KDO8PS is evenly divided between metal and non-metal forms, both having essentially identical structures. Mutagenesis and crystallographic studies suggest that one or two residues at most determine whether or not KDO8PS requires a metal for function, a clear example of "minimalist evolution". Quantum mechanical/molecular mechanical (QM/MM) simulations of both the enzymatic and non-enzymatic synthesis of KDO8P have revealed the mechanism underlying the switch between metal and non-metal dependent catalysis. The principle emerging from these studies is that this conversion is possible in KDO8PS because the metal is not involved in an activation process, but primarily contributes to orienting properly the reactants to lower the activation energy, an action easily mimicked by amino acid side-chains.

Fig. 1. KDO8P synthesis from PEP, A5P, and water

RS is the reactant state. Two stepwise mechanisms are shown requiring the formation of a transient carbanion (C_ION, upper path) or a transient zwitterion (Z_ION, lower path). Both paths converge to a linear intermediate (INT) that breaks down into KDO8P and P_i.

Fig. 2. Active site of metallo and non-metallo KDO8PS

A. Crystal structure of the active site of wild-type (metallo) and C11N (non-metallo) Aa. KDO8PS in a pre-RS state in which only PEP is bound. The wild type and the C11N mutant are shown with solid and transparent atoms/bonds, respectively. The active site metal in the wild-type enzyme is shown as a light-blue sphere. The water molecule (WAT) bound to the metal or hydrogen bonded to Asn11 is shown as a small red sphere. B,C. QM/MM optimized active sites of the wild-type (panel B) and C11N (panel C) Aa. KDO8PS in the reactant state (PEP + A5P + WAT). PEP is shown with cyan bonds, A5P with green bonds. The metal and its protein coordination are shown as a light-blue sphere and dashed lines. Yellow dashed lines highlight key hydrogen bonds that stabilize the relative positions of the substrates. Note the new position of the water molecule (WAT in panels B and C) displaced by the binding of A5P. There is a strong hydrogen bond between this water and the phosphate moiety of PEP, which is expected to favor the formation of the hydroxide ion that attacks C2^PEP. Also, in the absence of metal the protonated form of His185 (one of the metal ligands) is strongly stabilized with respect to its neutral counterpart (Panel C).

Fig. 3. PESs of the non-enzymatic condensation of PEP and A5P in water

The reactants (PEP + A5P + WAT with/without Zn²⁺/acetamide) were immersed in a sphere of explicit waters of 26 Å radius. A5P, PEP, the single water molecule involved in the reaction, and the metal ion or the acetamide molecule were treated quantum mechanically. All the other water molecules were treated with a molecular mechanics force field. Each PES is defined by two reaction coordinates: formation of the bond between C3^PEP and C1^A5P, and formation of the bond between the oxygen of water and C2^PEP. A. PES I: no Zn²⁺ or acetamide. B. PES II: at RS Zn²⁺ is coordinated to the water molecule that attacks C2^PEP; at TS1 and TS2 Zn²⁺ is coordinated to the same water molecule and to A5P carbonyl oxygen. C. PES III: Zn²⁺ is coordinated to A5P carbonyl oxygen. D. PES IV: acetamide (mimicking an asparagine side chain) is hydrogen bonded to A5P carbonyl oxygen. CC and CO distances are in Å, QM/MM energies are in kcal/mol. Colors on the PESs and on the projected contours below the PESs reflect QM/MM energy levels, as represented in the reference bar on the side.

Fig. 4. Transition states in the non-enzymatic condensation of PEP and A5P in water

The relative positions of PEP, A5P, Z_ION, Zn²⁺, water, and acetamide at TS1 (top row) and TS2 (bottom row) are shown for the PESs (I to IV, from left to right) of Figure 3. PEP is shown with cyan bonds, A5P with green bonds, Z_ION with purple bonds, acetamide with white bonds. Zn²⁺ is shown as a light-blue sphere. Hydrogen bonds are shown as yellow dashed lines. Zn²⁺ coordination is shown as blue dashed lines. Red dashed lines represent the C3^PEP–C1^A5P bond (TS1) or the O^WAT–C2^Z_ION bond (TS2) and reflect a pure bond-stretching mode corresponding to the single imaginary frequency identified at TS1 and TS2. Distances in Angstroms are shown next to each line.

Fig. 5. QM/MM optimized active sites of wild-type and C11N Aa. KDO8PS with the reaction intermediate INT bound

A, wild-type; B. C11N Aa. KDO8PS. INT is shown with slate bonds. The corresponding reactant states, as already shown in Figures 2B and 2C, are superimposed and represented with transparent atoms/bonds. Note the almost complete absence of motion in the protein between the RS and INT states.