Catalytic Cycle of Neisseria meningitidis CMP-Sialic Acid Synthetase Illustrated by High-Resolution Protein Crystallography

Abstract

Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of α-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the α-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

Figure 1.

NmCSS homodimer structure. (A) Structure of the ligand-free NmCSS homodimer. For contrast, one monomer is shown in bronze and the other in the rainbow spectrum starting with blue at the N-terminus and ending with red at the C-terminus. Each monomer consists of a globular nucleotide-binding domain and an extended dimerization domain where it interacts with its partner monomer. β-Sheets are labeled with numbers and α-helices with letters, with the exception of the 3₁₀ helix located in the dimerization domain of each monomer. (B) Structure of NmCSS homodimer with ligands CMP and Neu5Ac2en bound in the active site, drawn as sticks with yellow- and green-colored carbon atoms, respectively. Citrate binding at dimer interface is drawn in sticks with white-colored carbon atoms.

Figure 2.

Binding orientation of CTP in the active site. CTP (white carbons) bound in the A subunit of the dimer with two calcium ions (green spheres) ligated to the phosphate moieties. Calcium ions (A and B) work together with the P-loop (residues 12–22) to properly position and stabilize the negative charge on the triphosphate group. Calcium ion A ligates to the α-phosphate, Asp211, and four water molecules (red spheres). Asp209, which was observed to adopt two conformations with one binding to the Ca²⁺(A) water ligands, together with Asp211 make up the conserved DXD motif. Calcium ion B ligates to all three phosphates of CTP together with four water molecules. Calcium ligations are highlighted with yellow dashed lines, and hydrogen bonds between the protein and Ca-CTP are magenta in color, while the P-loop stabilization interaction between Arg12-Asp78-Lys16 is drawn with green dashed lines.

Figure 3.

Mechanism of active site closing. (A) After CTP and Ca²⁺ bind to monomer A, the enzyme maintains in the open state until the arrival of sialic acid. (B) As Neu5Ac2en binds to the nucleotide-binding (NB) domain of monomer A (white), interactions involving 5 residues from the dimerization domain of monomer B (brown) and sialic acid (yellow sticks) cause the distance between dimerization and NB domain to decrease by up to 8 Å. (C) After formation of product, contacts between the dimerization domain and sialic acid are broken, and the active site is reopened. (D) Superposition of nucleotide-binding domains of the A monomers from the CTP-bound structure (green) onto the CMP/Neu5Ac2en structure (yellow). Relative orientation of the dimerization domain rotates closed upon binding Neu5Ac2en (highlighted by arrow).

Figure 4.

Ligands involved in dimer closing. (A) The sialic acid analogue (yellow) has numerous contacts with both the nucleotide-binding domain of monomer A (white sticks) and the dimerization domain of monomer B (brown sticks). Three ordered water molecules indicate a solvent pocket, which may allow for substrate variation. (B) Summary of contacts between NmCSS and Neu5Ac2en.

Figure 5.

Product-bound open state of the NmCSS active site. After CMP-Neu5Ac product formation (green-colored bonds), the enzyme releases pyrophosphate and Ca²⁺(B) and leaves behind Ca²⁺(A) and product. Interactions between sialic acid and the nucleotide-binding domain are maintained, but those between sialic acid and the dimerization domain of the opposite monomer are broken.

Figure 6.

Comparison of NmCSS to similar enzymes. Interactions between protein and sugar are white dashes. Those between protein and nucleotide are yellow. (A) NmCSS/CMP/Neu5Ac2en with an overlay of CTP (sticks) from the NmCSS/CTP structure, highlighting residues from an opposite monomer, which interact with sialic acid and possibly with CTP. Panels B and C focus on only a small selection of relevant protein–sugar interactions for comparison to Lys142 and Arg165 of NmCSS. (B) EcKdsB/CTP/Kdo2en (PDB ID 3K8D). Kdo2en is a nonreacting analogue of KdsB’s natural substrate. (C) MmCSS/CMP-Neu5Ac (PDB ID 1QWJ). A bridging arginine between the dimerization domain and substrate of the opposite monomer is a common feature.

Figure 7.

Proposed mechanism of NmCSS. (A) Model of the NmCSS active site in the presence of both substrates. The conformation of CTP (green sticks) in the presence of Ca²⁺(A) and Ca²⁺(B) (gray spheres) superimposed into the NmCSS/CMP/Neu5Ac2en active site, in which Neu5Ac2en has been replaced with Neu5Ac suggests a clash (red dashes) between Ca²⁺(A) and Arg165 (thin brown sticks). However, a different rotamer of Arg165 (thick brown sticks) would allow for the observed Ca²⁺(A) placement while preserving the negative-charge stabilization of CTP’s γ-phosphate by the arginine guanidinium group. (B) (i) CTP binds to the nucleotide binding (NB) domain first in addition to two divalent cations. (ii) Next, Neu5Ac enters the active site and associates initially with the NB domain but quickly (iii) draws together the NB and dimerization domains to form the closed state (all residues involved in Neu5Ac binding are summarized below the structure for simplicity. (iv) Divalent cation M²⁺ coordinates the C2-OH, allowing a water molecule to deprotonate C2-OH long enough for the hydroxyl O to attack the α-phosphate. (v) After product formation, pyrophosphate (PP_i) and M²⁺(B) are released from the active site and the active site reopens.

Scheme 1.

Reaction Catalyzed by CMP-Sialic Acid Synthetases (CSSs)