Ceramidase

Function

Figure 1 Proposed mechanisms of the zinc-dependent hydrolysis of C2-ceramide (Black arrows) and the zinc-dependent synthesis of C2-ceramide from palmitate and sphingosine (Red arrows) by CerN . Figure adapted from Inoe et al.(2009)^[1]

CerN is an enzyme that catalyzes the cleavage of the Sphingolipid at the , producing .^{[2] [1]} As a neutral ceramidase, optimal catalytic activity of CerN occurs between pH 6.5-8.5.^[2] CerN cleaves the N-acyl linkage within ceramides via zinc-dependent hydrolysis and the enzyme is also capable of synthesizing ceramide from sphingosine and palmitic acid by the reverse mechanism.^[1][3] The zinc ion within the is coordinated by His97, His204, Glu411, Tyr448, and a water molecule. His97 and Tyr448 are required for zinc binding within the active site. Ligand binding within the active site is recognized by Gly25, His99, Arg160, and Tyr460.^[1] Ser27 and Gly25 stabilize ceramide within the active site by forming a water-mediated hydrogen bond with the central OH of ceramide, and the carbonyl oxygen is stabilized by the Tyr448 and Tyr460. Upon ligand binding, CerN enters the conformation. ^[1] His99 and Arg160 function in the catalysis of ceramide hydrolysis, as they deprotonate their coordinated water molecule to produce a hydroxide ion.^[1] The carbonyl carbon of ceramide undergoes a nucleophilic attack by the hydroxide ion (Figure 1).^[1] The carbonyl oxygen stabilized by Tyr448 and Tyr460 is then passed to the zinc ion, allowing for the breakage of the N-acyl linkage.^[1] Sphingosine is then released from the active site while the fatty acid remains bound to the zinc ion until it is replaced by a new water molecule, shifting CerN into the conformation.^[1] The synthesis of ceramide from palmitate and sphingosine occurs via the same mechanism but in reverse (Figure 1). ^[1]

Refs ^[2], ^[1] , ^[3]

Structural highlights

CerN consists of two domains: a .^[1] Three β-sheets, each formed from four β-strands, compose a β-prism fold at the center of the N-terminal domain.^[1] Surrounding the β-prism fold are 11 α-helices, forming an .^[1] The immunoglobulin-like C-terminal domain is composed of two β-sheets, containing four β-strands each, forming a .^[1] Between the N- and C-terminal domains is a magnesium/calcium ion binding site that links together the two domains.^[1] His37, Asp579, Asp581, and Thr854 interact with divalent cations within the .^[1] A is located within the N-terminal domain active site, where it is coordinated by His97, His204, Glu411, and a water molecule.^[1]

Role of Ceramide and CerN in Bacterial Infections

Sphingolipids play key role in eukaryotic cell membrane structure and function.^[4][5] Additionally, sphingolipids act as signaling molecules for eukaryotic processes such as proliferation, apoptosis, inflammation, cell migration, and pathogen defense.^[5] Ceramide is considered as the central molecule in sphingolipid metabolism, as it can be converted to more complex sphingolipids or be broken down for the production of sphingosine and sphingosine-1-phosphate.^{[6] [7] [8]} Eukaryotes utilize ceramide in the formation of lipid rafts, lipid-protein platforms that alter the biophysical properties of cell membranes as well as localize receptors for signal amplification.^{[5] [7]} Sphingosine and sphingosine-1-phosphate, the products of ceramide catabolism, are important for pathogen defense by functioning as a potent anti-microbial and activating signal for host-immune responses, respectively.^[6]

Due to its abundance in eukaryotic cell membranes and role in pathogen defense, pathogens have evolved mechanisms to use host ceramides for their own pathogenesis.^[7] Ceramide-rich membrane platforms on human-epithelial cells serve as sites for the attachment and invasion of bacterial pathogens such as Neisseria gonorrhoeae.^[7] Pseudomonas aeruginosa is capable of detecting host-derived sphingosine, resulting in activation of P. aeruginosa sphingosine-responsive genes.^[9] The products of P. aeruginosa sphingosine-responsive genes are used for the detoxification of sphingosine, as well as its production from other host-derived sphingolipids, making P. aeruginosa sphingosine tolerant.^[9] Loss of P. aeruginosa sphingosine-responsive genes results in the inability of the bacteria to survive in the presence of sphingosine in vitro or in the murine lung.^[9] CerN is one of the proteins encoded by P. aeruginosa sphingosine-responsive genes used for the production of sphingosine from ceramide, with the added ability of functioning as a ceramide synthase.^[2][3][9] It has been hypothesized that the hydrolysis of host-membrane ceramide via CerN facilitates P. aeruginosa intracellular invasion, similar to other bacterial pathogens.^[2] CerN is also involved in P. aeruginosa biofilm production, a major virulence trait of the pathogen.^[10] Sphingosine production via CerN-mediated hydrolysis of host ceramide induces a biofilm formation at sites of infection, biofilm accumulation yields more ceramide hydrolysis, creating a positive-feedback loop for P. aeruginosa virulence.^[10]

Relevance

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