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A gatekeeper helix determines the substrate specificity of Sjögren-Larsson Syndrome enzyme fatty aldehyde dehydrogenase.

Keller MA, Zander U, Fuchs JE, Kreutz C, Watschinger K, Mueller T, Golderer G, Liedl KR, Ralser M, Kräutler B, Werner ER, Marquez JA - Nat Commun (2014)

Bottom Line: Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase.The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases.Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria [2] Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis court Rd, Cambridge CB2 1GA, UK.

ABSTRACT
Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren-Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a 'gatekeeper' helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

No MeSH data available.


Related in: MedlinePlus

A proposed reaction mechanism for FALDH.(a) Reaction schemes for the oxidation of long-chain fatty aldehydes by FALDH. (1) Cys-241 is activated by a general base (possible active residues are (a) Glu-207 or (b) Glu-331), and then initiates a nucleophilic attack on the carbonyl carbon of the aldehyde. Correct positioning of the polar aldehyde head group is supported by Asn-112. (2) The formed oxyanion collapses, eliminating a hydride ion, which is transferred to NAD. (3) A proton is abstracted from a water molecule, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate. (4) A recurrent oxyanion collapse terminates the thiohemiacetal bond and releases the fatty acid product. (b) Influence of mutated active site residues on enzymatic capacity. Mutations where introduced by site-directed mutagenesis and correct folding of the purified enzymes was verified (see Methods). N112A, E207Q, C241S and E331Q mutations completely abolished all measurable enzymatic activity. Y113F had now significant effect on enzymatic activity. Exchanging the previously reported catalytic amino acid Y410 with phenylalanine had only small impact on the FALDH reaction. Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (c) Stereoselectivity of the FALDH reaction: 1H NMR spectroscopy was used to monitor the stereospecific collapse of the NAD C4-H splitting pattern (C4-Hpro-S: 2.77; C4-Hpro-R: 2.87 p.p.m.) during the enzymatic hydride transfer from deuterated substrates to the cofactor (spectra after 60 min of reaction time are shown). As control, we used the pro-R-specific horse liver alcohol dehydrogenase and 1,2-[D]-ethanol. For the closely related ALDH3A1, a special NAD-binding mode with a presumed pro-S stereoselectivity was reported. In contrast, for FALDH, we exclusively observe the presence of the downfield C4-Hpro-S signal (2.77 p.p.m.), indicating a specific deuteration of the pro-R position. Additional controls for this experiment are depicted in Supplementary Fig. 4b.
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f4: A proposed reaction mechanism for FALDH.(a) Reaction schemes for the oxidation of long-chain fatty aldehydes by FALDH. (1) Cys-241 is activated by a general base (possible active residues are (a) Glu-207 or (b) Glu-331), and then initiates a nucleophilic attack on the carbonyl carbon of the aldehyde. Correct positioning of the polar aldehyde head group is supported by Asn-112. (2) The formed oxyanion collapses, eliminating a hydride ion, which is transferred to NAD. (3) A proton is abstracted from a water molecule, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate. (4) A recurrent oxyanion collapse terminates the thiohemiacetal bond and releases the fatty acid product. (b) Influence of mutated active site residues on enzymatic capacity. Mutations where introduced by site-directed mutagenesis and correct folding of the purified enzymes was verified (see Methods). N112A, E207Q, C241S and E331Q mutations completely abolished all measurable enzymatic activity. Y113F had now significant effect on enzymatic activity. Exchanging the previously reported catalytic amino acid Y410 with phenylalanine had only small impact on the FALDH reaction. Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (c) Stereoselectivity of the FALDH reaction: 1H NMR spectroscopy was used to monitor the stereospecific collapse of the NAD C4-H splitting pattern (C4-Hpro-S: 2.77; C4-Hpro-R: 2.87 p.p.m.) during the enzymatic hydride transfer from deuterated substrates to the cofactor (spectra after 60 min of reaction time are shown). As control, we used the pro-R-specific horse liver alcohol dehydrogenase and 1,2-[D]-ethanol. For the closely related ALDH3A1, a special NAD-binding mode with a presumed pro-S stereoselectivity was reported. In contrast, for FALDH, we exclusively observe the presence of the downfield C4-Hpro-S signal (2.77 p.p.m.), indicating a specific deuteration of the pro-R position. Additional controls for this experiment are depicted in Supplementary Fig. 4b.

Mentions: The reaction mechanism for FALDH has been proposed to be based on a nucleophilic attack of the fatty aldehyde substrate by Cys-241. Lloyd et al.19 speculate that the resultant thiohemiacetal is deprotonated by Tyr-410, which initiates a hydride transfer from the substrate to the NAD cofactor. Glu-207 would then mediate a nucleophilic attack of a hydroxide anion, and the fatty acid product is eventually released (Fig. 4a). To investigate the effect of the FALDH reaction mechanism on substrate specificity, we performed site-directed mutagenesis of residues involved in catalysis (C241S, E207Q and Y410F). An additional set of conserved residues was selected based on their proximity to the active site in the crystal structure, or was selected based on published evidence for their catalytic function in other ALDH isozymes (E331Q, N112A and Y113F). Mutant FALDH variants were cloned, purified and their correct folding was verified by circular dichroism measurements. A fluorometric assay was used to determine the enzymatic activity of purified wild-type and mutated FALDH proteins (see Methods). Three medium to long aliphatic chain fatty aldehydes, octadecanal, dodecanal and hexadecanal were used as substrates. Improper recycling of hexadecanal derived from sphingosine-1-phosphate catabolism and other fatty aldehydes originating from other sources has been proposed to be a major cause for SLS3. Enzymatic assays demonstrated that, as expected, Cys-241 and Glu-207 play a critical role in catalysis, since the corresponding mutants showed no activity against any of the substrates (Fig. 4b). The Y410F mutant showed normal Vmax/KM levels against octanal and dodecanal and a somewhat reduced but still considerable catalytic capacity for hexadecanal. Hence, Tyr-410 is not essential for FALDH activity, but may be required for efficient substrate turnover. The enzymatic assays also revealed important roles for Glu-331 and Asn-112: proteins mutant for E331Q and N112A did not exhibit enzymatic activity in our assay. In contrast, mutating Tyr-113 had no effect on catalysis (Fig. 4b).


A gatekeeper helix determines the substrate specificity of Sjögren-Larsson Syndrome enzyme fatty aldehyde dehydrogenase.

Keller MA, Zander U, Fuchs JE, Kreutz C, Watschinger K, Mueller T, Golderer G, Liedl KR, Ralser M, Kräutler B, Werner ER, Marquez JA - Nat Commun (2014)

A proposed reaction mechanism for FALDH.(a) Reaction schemes for the oxidation of long-chain fatty aldehydes by FALDH. (1) Cys-241 is activated by a general base (possible active residues are (a) Glu-207 or (b) Glu-331), and then initiates a nucleophilic attack on the carbonyl carbon of the aldehyde. Correct positioning of the polar aldehyde head group is supported by Asn-112. (2) The formed oxyanion collapses, eliminating a hydride ion, which is transferred to NAD. (3) A proton is abstracted from a water molecule, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate. (4) A recurrent oxyanion collapse terminates the thiohemiacetal bond and releases the fatty acid product. (b) Influence of mutated active site residues on enzymatic capacity. Mutations where introduced by site-directed mutagenesis and correct folding of the purified enzymes was verified (see Methods). N112A, E207Q, C241S and E331Q mutations completely abolished all measurable enzymatic activity. Y113F had now significant effect on enzymatic activity. Exchanging the previously reported catalytic amino acid Y410 with phenylalanine had only small impact on the FALDH reaction. Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (c) Stereoselectivity of the FALDH reaction: 1H NMR spectroscopy was used to monitor the stereospecific collapse of the NAD C4-H splitting pattern (C4-Hpro-S: 2.77; C4-Hpro-R: 2.87 p.p.m.) during the enzymatic hydride transfer from deuterated substrates to the cofactor (spectra after 60 min of reaction time are shown). As control, we used the pro-R-specific horse liver alcohol dehydrogenase and 1,2-[D]-ethanol. For the closely related ALDH3A1, a special NAD-binding mode with a presumed pro-S stereoselectivity was reported. In contrast, for FALDH, we exclusively observe the presence of the downfield C4-Hpro-S signal (2.77 p.p.m.), indicating a specific deuteration of the pro-R position. Additional controls for this experiment are depicted in Supplementary Fig. 4b.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4109017&req=5

f4: A proposed reaction mechanism for FALDH.(a) Reaction schemes for the oxidation of long-chain fatty aldehydes by FALDH. (1) Cys-241 is activated by a general base (possible active residues are (a) Glu-207 or (b) Glu-331), and then initiates a nucleophilic attack on the carbonyl carbon of the aldehyde. Correct positioning of the polar aldehyde head group is supported by Asn-112. (2) The formed oxyanion collapses, eliminating a hydride ion, which is transferred to NAD. (3) A proton is abstracted from a water molecule, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate. (4) A recurrent oxyanion collapse terminates the thiohemiacetal bond and releases the fatty acid product. (b) Influence of mutated active site residues on enzymatic capacity. Mutations where introduced by site-directed mutagenesis and correct folding of the purified enzymes was verified (see Methods). N112A, E207Q, C241S and E331Q mutations completely abolished all measurable enzymatic activity. Y113F had now significant effect on enzymatic activity. Exchanging the previously reported catalytic amino acid Y410 with phenylalanine had only small impact on the FALDH reaction. Data are shown as mean±s.e.m. (calculated with error propagation, n=3). (c) Stereoselectivity of the FALDH reaction: 1H NMR spectroscopy was used to monitor the stereospecific collapse of the NAD C4-H splitting pattern (C4-Hpro-S: 2.77; C4-Hpro-R: 2.87 p.p.m.) during the enzymatic hydride transfer from deuterated substrates to the cofactor (spectra after 60 min of reaction time are shown). As control, we used the pro-R-specific horse liver alcohol dehydrogenase and 1,2-[D]-ethanol. For the closely related ALDH3A1, a special NAD-binding mode with a presumed pro-S stereoselectivity was reported. In contrast, for FALDH, we exclusively observe the presence of the downfield C4-Hpro-S signal (2.77 p.p.m.), indicating a specific deuteration of the pro-R position. Additional controls for this experiment are depicted in Supplementary Fig. 4b.
Mentions: The reaction mechanism for FALDH has been proposed to be based on a nucleophilic attack of the fatty aldehyde substrate by Cys-241. Lloyd et al.19 speculate that the resultant thiohemiacetal is deprotonated by Tyr-410, which initiates a hydride transfer from the substrate to the NAD cofactor. Glu-207 would then mediate a nucleophilic attack of a hydroxide anion, and the fatty acid product is eventually released (Fig. 4a). To investigate the effect of the FALDH reaction mechanism on substrate specificity, we performed site-directed mutagenesis of residues involved in catalysis (C241S, E207Q and Y410F). An additional set of conserved residues was selected based on their proximity to the active site in the crystal structure, or was selected based on published evidence for their catalytic function in other ALDH isozymes (E331Q, N112A and Y113F). Mutant FALDH variants were cloned, purified and their correct folding was verified by circular dichroism measurements. A fluorometric assay was used to determine the enzymatic activity of purified wild-type and mutated FALDH proteins (see Methods). Three medium to long aliphatic chain fatty aldehydes, octadecanal, dodecanal and hexadecanal were used as substrates. Improper recycling of hexadecanal derived from sphingosine-1-phosphate catabolism and other fatty aldehydes originating from other sources has been proposed to be a major cause for SLS3. Enzymatic assays demonstrated that, as expected, Cys-241 and Glu-207 play a critical role in catalysis, since the corresponding mutants showed no activity against any of the substrates (Fig. 4b). The Y410F mutant showed normal Vmax/KM levels against octanal and dodecanal and a somewhat reduced but still considerable catalytic capacity for hexadecanal. Hence, Tyr-410 is not essential for FALDH activity, but may be required for efficient substrate turnover. The enzymatic assays also revealed important roles for Glu-331 and Asn-112: proteins mutant for E331Q and N112A did not exhibit enzymatic activity in our assay. In contrast, mutating Tyr-113 had no effect on catalysis (Fig. 4b).

Bottom Line: Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase.The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases.Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria [2] Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis court Rd, Cambridge CB2 1GA, UK.

ABSTRACT
Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren-Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a 'gatekeeper' helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations.

No MeSH data available.


Related in: MedlinePlus