Ceramidase: Difference between revisions

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[[Image:CerN Mechanism Okino2009.jpg|300px|right|thumb|'''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)<ref name="Inoue">PMID:19088069</ref>]]

'''CerN''' is an enzyme that catalyzes the cleavage of the [https://en.wikipedia.org/wiki/Sphingolipid Sphingolipid] <scene name='91/910024/Ceramide/3'>Ceramide</scene> at the <scene name='91/910024/Ceramide18/1'>N-acyl linkage</scene>, producing <scene name='91/910024/Ceramide/4'>sphingosine and a fatty acid</scene>.<ref name="Okino">PMID:9603946</ref> <ref name="Inoue">PMID:19088069</ref> As a neutral ceramidase, optimal catalytic activity of CerN occurs between pH 6.5-8.5.<ref name="Okino">PMID:9603946</ref> 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.<ref name="Inoue">PMID:19088069</ref><ref name="Reverse">PMID:10832092</ref> The zinc ion within the <scene name='91/910024/Activesite2/3'>active site</scene> 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''.<ref name="Inoue">PMID:19088069</ref> 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 <scene name='91/910024/Closedsurf_use/1'>closed</scene> conformation. <ref name="Inoue">PMID:19088069</ref> ''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.<ref name="Inoue">PMID:19088069</ref> 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 <scene name='91/910024/Activesite_open_surf/2'>open</scene> conformation. The synthesis of ceramide from palmitate and sphingosine occurs via the same mechanism but in reverse ('''Figure 1'''). <ref name="Inoue">PMID:19088069</ref>

Upon ligand binding, CerN enters the <scene name='91/910024/Closedsurf_use/1'>closed</scene> conformation. <ref name="Inoue">PMID:19088069</ref> ''His99 and Arg160 function in the catalysis of ceramide hydrolysis'', as they deprotonate their coordinated water molecule to produce a hydroxide ion.<ref name="Inoue">PMID:19088069</ref> The carbonyl carbon of ceramide undergoes a '''nucleophilic attack''' by the hydroxide ion ('''Figure 1''').<ref name="Inoue">PMID:19088069</ref> The carbonyl oxygen stabilized by Tyr448 and Tyr460 is then passed to the zinc ion, allowing for the breakage of the N-acyl linkage.<ref name="Inoue">PMID:19088069</ref> 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 <scene name='91/910024/Activesite_open_surf/2'>open</scene> conformation.<ref name="Inoue">PMID:19088069</ref> The synthesis of ceramide from palmitate and sphingosine occurs via the same mechanism but in reverse ('''Figure 1'''). <ref name="Inoue">PMID:19088069</ref>

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Refs <ref name="Okino">PMID:9603946</ref>, <ref name="Inoue">PMID:19088069</ref> , <ref name="Reverse">PMID:10832092</ref>

== 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 <scene name='91/910024/Bprism/3'>α+ β 2-layer sandwich fold</scene>. The immunoglobulin-like C-terminal domain is composed of two β-sheets, containing four β-strands each, forming a <scene name='91/910024/Igfold/1'>β-sandwich fold</scene>. 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 <scene name='91/910024/Mg_bs/1'>magnesium/calcium ion binding site</scene>. A <scene name='91/910024/Zinc_bs/3'>second metal-binding site containing a zinc ion</scene> is located within the N-terminal domain active site, where it is coordinated by His97, His204, Glu411, and a water molecule.

CerN consists of two domains: a catalytic domain near the N-terminal and an immunoglobulin-fold domain near the C-terminal.<ref name="Inoue">PMID:19088069</ref> Three β-sheets, each formed from four β-strands, compose a β-prism fold at the center of the N-terminal domain.<ref name="Inoue">PMID:19088069</ref> Surrounding the β-prism fold are 11 α-helices, forming an <scene name='91/910024/Bprism/3'>α+ β 2-layer sandwich fold</scene>.<ref name="Inoue">PMID:19088069</ref> The immunoglobulin-like C-terminal domain is composed of two β-sheets, containing four β-strands each, forming a <scene name='91/910024/Igfold/1'>β-sandwich fold</scene>.<ref name="Inoue">PMID:19088069</ref> Between the N- and C-terminal domains is a magnesium/calcium ion binding site that links together the two domains.<ref name="Inoue">PMID:19088069</ref> His37, Asp579, Asp581, and Thr854 interact with divalent cations within the <scene name='91/910024/Mg_bs/1'>magnesium/calcium ion binding site</scene>.<ref name="Inoue">PMID:19088069</ref> A <scene name='91/910024/Zinc_bs/3'>second metal-binding site containing a zinc ion</scene> is located within the N-terminal domain active site, where it is coordinated by His97, His204, Glu411, and a water molecule.<ref name="Inoue">PMID:19088069</ref>

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@@ Line 3: / Line 3: @@
 [[Image:CerN Mechanism Okino2009.jpg|300px|right|thumb|'''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)<ref name="Inoue">PMID:19088069</ref>]]
 '''CerN''' is an enzyme that catalyzes the cleavage of the [https://en.wikipedia.org/wiki/Sphingolipid Sphingolipid] <scene name='91/910024/Ceramide/3'>Ceramide</scene> at the <scene name='91/910024/Ceramide18/1'>N-acyl linkage</scene>, producing <scene name='91/910024/Ceramide/4'>sphingosine and a fatty acid</scene>.<ref name="Okino">PMID:9603946</ref> <ref name="Inoue">PMID:19088069</ref> As a neutral ceramidase, optimal catalytic activity of CerN occurs between pH 6.5-8.5.<ref name="Okino">PMID:9603946</ref> 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.<ref name="Inoue">PMID:19088069</ref><ref name="Reverse">PMID:10832092</ref> The zinc ion within the <scene name='91/910024/Activesite2/3'>active site</scene> 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''.<ref name="Inoue">PMID:19088069</ref> 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 <scene name='91/910024/Closedsurf_use/1'>closed</scene> conformation. <ref name="Inoue">PMID:19088069</ref> ''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.<ref name="Inoue">PMID:19088069</ref> 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 <scene name='91/910024/Activesite_open_surf/2'>open</scene> conformation. The synthesis of ceramide from palmitate and sphingosine occurs via the same mechanism but in reverse ('''Figure 1'''). <ref name="Inoue">PMID:19088069</ref>
+Upon ligand binding, CerN enters the <scene name='91/910024/Closedsurf_use/1'>closed</scene> conformation. <ref name="Inoue">PMID:19088069</ref> ''His99 and Arg160 function in the catalysis of ceramide hydrolysis'', as they deprotonate their coordinated water molecule to produce a hydroxide ion.<ref name="Inoue">PMID:19088069</ref> The carbonyl carbon of ceramide undergoes a '''nucleophilic attack''' by the hydroxide ion ('''Figure 1''').<ref name="Inoue">PMID:19088069</ref> The carbonyl oxygen stabilized by Tyr448 and Tyr460 is then passed to the zinc ion, allowing for the breakage of the N-acyl linkage.<ref name="Inoue">PMID:19088069</ref> 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 <scene name='91/910024/Activesite_open_surf/2'>open</scene> conformation.<ref name="Inoue">PMID:19088069</ref> The synthesis of ceramide from palmitate and sphingosine occurs via the same mechanism but in reverse ('''Figure 1'''). <ref name="Inoue">PMID:19088069</ref>
@@ Line 9: / Line 9: @@
 Refs <ref name="Okino">PMID:9603946</ref>, <ref name="Inoue">PMID:19088069</ref> , <ref name="Reverse">PMID:10832092</ref>
 == 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 <scene name='91/910024/Bprism/3'>α+ β 2-layer sandwich fold</scene>. The immunoglobulin-like C-terminal domain is composed of two β-sheets, containing four β-strands each, forming a <scene name='91/910024/Igfold/1'>β-sandwich fold</scene>. 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 <scene name='91/910024/Mg_bs/1'>magnesium/calcium ion binding site</scene>. A <scene name='91/910024/Zinc_bs/3'>second metal-binding site containing a zinc ion</scene> is located within the N-terminal domain active site, where it is coordinated by His97, His204, Glu411, and a water molecule.
+CerN consists of two domains: a catalytic domain near the N-terminal and an immunoglobulin-fold domain near the C-terminal.<ref name="Inoue">PMID:19088069</ref> Three β-sheets, each formed from four β-strands, compose a β-prism fold at the center of the N-terminal domain.<ref name="Inoue">PMID:19088069</ref> Surrounding the β-prism fold are 11 α-helices, forming an <scene name='91/910024/Bprism/3'>α+ β 2-layer sandwich fold</scene>.<ref name="Inoue">PMID:19088069</ref> The immunoglobulin-like C-terminal domain is composed of two β-sheets, containing four β-strands each, forming a <scene name='91/910024/Igfold/1'>β-sandwich fold</scene>.<ref name="Inoue">PMID:19088069</ref> Between the N- and C-terminal domains is a magnesium/calcium ion binding site that links together the two domains.<ref name="Inoue">PMID:19088069</ref> His37, Asp579, Asp581, and Thr854 interact with divalent cations within the <scene name='91/910024/Mg_bs/1'>magnesium/calcium ion binding site</scene>.<ref name="Inoue">PMID:19088069</ref> A <scene name='91/910024/Zinc_bs/3'>second metal-binding site containing a zinc ion</scene> is located within the N-terminal domain active site, where it is coordinated by His97, His204, Glu411, and a water molecule.<ref name="Inoue">PMID:19088069</ref>