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. The carbonyl carbon of ceramide undergoes a nucleophilic attack by the hydroxide ion (Figure 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. 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 catalytic domain near the N-terminal and an immunoglobulin-fold domain near the C-terminal. Three β-sheets, each formed from four β-strands, compose a β-prism fold at the center of the N-terminal domain. Surrounding the β-prism fold are 11 α-helices, forming an α+ β 2-layer sandwich fold. The immunoglobulin C-terminal domain is composed of two β-sheets, containing four β-strands each, forming a β-sandwich fold. Between the N- and C-terminal domains is a magnesium/calcium ion binding site that links together the two domains. His37, Asp579, Asp581, and Thr854 interact with divalent cations within the magnesium/calcium ion binding site. A second metal-binding site containing a zinc ion is located within the N-terminal domain active site, where it is coordinated by His97, His204, Glu411, and a water molecule.

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Disease

p^[2]

Relevance

p