Structural basis for substrate specificity and mechanism of N-acetyl-D-neuraminic acid lyase from Pasteurella multocida

Abstract

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac, the most common form of sialic acid) to form pyruvate and N-acetyl-d-mannosamine. Although equilibrium favors sialic acid cleavage, these enzymes can be used for high-yield chemoenzymatic synthesis of structurally diverse sialic acids in the presence of excess pyruvate. Engineering these enzymes to synthesize structurally modified natural sialic acids and their non-natural derivatives holds promise in creating novel therapeutic agents. Atomic-resolution structures of these enzymes will greatly assist in guiding mutagenic and modeling studies to engineer enzymes with altered substrate specificity. We report here the crystal structures of wild-type Pasteurella multocida N-acetylneuraminate lyase and its K164A mutant. Like other bacterial lyases, it assembles into a homotetramer with each monomer folding into a classic (β/α)₈ TIM barrel. Two wild-type structures were determined, in the absence of substrates, and trapped in a Schiff base intermediate between Lys164 and pyruvate, respectively. Three structures of the K164A variant were determined: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide. The structures reveal that every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme, and the residues that determine specificity were identified. Additionally, the structures provide some clues for explaining the natural discrimination of sialic acid substrates between the P. multocida and Escherichia coli NALs.

Figure 1

(A) PmNAL monomer. Ribbon drawing of wild-type PmNAL monomer represented by a rainbow color scheme from blue (N-terminus) to red (C-terminus). Each monomer consists of a TIM barrel (β/α)₈-fold followed by an additional three alpha-helices at the C-terminus (αI, αJ, αK). Secondary structure elements are labeled. Displayed in the active site is the Lys164-pyruvate Schiff base intermediate shown with sticks in yellow colored carbon atoms. (B) PmNAL tetramer with each subunit displayed in a different color. The tetramer possesses D2 symmetry with access to the four active sites from the central cavity.

Figure 1

(A) PmNAL monomer. Ribbon drawing of wild-type PmNAL monomer represented by a rainbow color scheme from blue (N-terminus) to red (C-terminus). Each monomer consists of a TIM barrel (β/α)₈-fold followed by an additional three alpha-helices at the C-terminus (αI, αJ, αK). Secondary structure elements are labeled. Displayed in the active site is the Lys164-pyruvate Schiff base intermediate shown with sticks in yellow colored carbon atoms. (B) PmNAL tetramer with each subunit displayed in a different color. The tetramer possesses D2 symmetry with access to the four active sites from the central cavity.

Figure 2

(A) Schiff base intermediate in the active site of PmNAL. The aldol reaction is mediated by conserved residue Tyr136, and an ordered water molecule, likely coming from the oxygen of the pyruvate, is also shown. The electron density represented by blue mesh contoured at 1σ showing continuous density linking C2 of pyruvate and Nζ of Lys164. (B) Hydrogen-bonding network around the pyruvate moiety forming a Schiff base with Lys164. Hydrogen-bond interactions between the Schiff base intermediate and nearby residues are represented by yellow dashed lines with the corresponding distances shown in Ångstroms. The carboxyl group of the pyruvate forms hydrogen bonds to the main-chain amide nitrogens of Ser47 and Thr48 as well as Oγ1 of Thr48. Two conformations of Phe189 are shown near the active site.

Figure 2

(A) Schiff base intermediate in the active site of PmNAL. The aldol reaction is mediated by conserved residue Tyr136, and an ordered water molecule, likely coming from the oxygen of the pyruvate, is also shown. The electron density represented by blue mesh contoured at 1σ showing continuous density linking C2 of pyruvate and Nζ of Lys164. (B) Hydrogen-bonding network around the pyruvate moiety forming a Schiff base with Lys164. Hydrogen-bond interactions between the Schiff base intermediate and nearby residues are represented by yellow dashed lines with the corresponding distances shown in Ångstroms. The carboxyl group of the pyruvate forms hydrogen bonds to the main-chain amide nitrogens of Ser47 and Thr48 as well as Oγ1 of Thr48. Two conformations of Phe189 are shown near the active site.

Figure 3

Active site of PmNAL K164A with sialic acids bound. (A) 2Fo-Fc electron density map contoured at 1σ is shown in blue mesh modeled around the Neu5Ac (sticks with white carbon bonds). (B) Active site showing potential hydrogen bonds between Neu5Ac and protein atoms. (C) 2Fo-Fc electron density map contoured at 1σ showing the configuration of Neu5Gc bound to PmNAL K164A. (D) Active site showing potential hydrogen bonds between Neu5Gc and enzyme.

Figure 4

Flatten two-dimensional representation of Interactions between PmNAL K164A and Neu5Ac. Covalent bonds are represented by solid green lines for Neu5Ac and tan lines for protein residues. Hydrogen bonds are shown as black dashed lines. van der Waals contacts are drawn as hashed red lines around an atom pointing in the direction of the corresponding interacting atom. Diagram was generated with the program LIGPLOT (48).

Figure 5

Comparison of active site pockets between PmNAL and EcNAL. Stereoview showing the wild-type PmNAL Schiff base complex (white), superposed onto the PmNAL K164 Neu5Ac complex (green) and the EcNAL (PDBID: 3LBC) structure (magenta). Phe189 in PmNAL takes on two conformations in the pyruvate-bound Schiff base, which might allow for modifications on O8 in the PmNAL enzyme. This residue is Tyrosine in the E. coli enzyme, which would likely not be able to accommodate similar multiple conformations due to the hydroxyl residue and the presence of Asn188 occupying the same pocket.