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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

An additional gatekeeper helix constrains substrate specificity by restricting the substrate binding funnel.(a) Cross section through the active site of FALDH. The NAD-binding pocket and the substrate funnel positioned on opposite sides of the enzyme and merge in its central active site. Active site residues are indicated in red (Asn-112, Glu-207, Cys-241 and Glu-331). The electron density found in the substrate funnel is indicated in yellow. The electron density mesh was generated at 1.2 sigma within 1.6 Å around a hexadecanoic acid molecule, which was previously modelled into the electron density. The gatekeeper helix, covering the substrate funnel entrance is shown in green. The substrate funnels of (b) FALDH and (c) ALDH3A1 (3sza) were characterized using the MOLE toolkit for PyMOL. While in FALDH, the additional gatekeeper helix (shown in green) creates a kink in the channel (shown in yellow), the wide, linear arrangement of the substrate funnel entrance of ALDH3A1 is not restricted.
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f3: An additional gatekeeper helix constrains substrate specificity by restricting the substrate binding funnel.(a) Cross section through the active site of FALDH. The NAD-binding pocket and the substrate funnel positioned on opposite sides of the enzyme and merge in its central active site. Active site residues are indicated in red (Asn-112, Glu-207, Cys-241 and Glu-331). The electron density found in the substrate funnel is indicated in yellow. The electron density mesh was generated at 1.2 sigma within 1.6 Å around a hexadecanoic acid molecule, which was previously modelled into the electron density. The gatekeeper helix, covering the substrate funnel entrance is shown in green. The substrate funnels of (b) FALDH and (c) ALDH3A1 (3sza) were characterized using the MOLE toolkit for PyMOL. While in FALDH, the additional gatekeeper helix (shown in green) creates a kink in the channel (shown in yellow), the wide, linear arrangement of the substrate funnel entrance of ALDH3A1 is not restricted.

Mentions: The catalytic and cofactor-binding domains of human FALDH (huFALDH) are structurally similar to that of cytosolic rat and human ALDH3A1 (with r.m.s.d. of 0.718 and 0.543, respectively) and contain all the structural features that characterize the ALDH family141620. The catalytic domain contains the catalytic cysteine (Cys-241, corresponding to Cys-302 in mammalian class 1 and class 2 ALDHs16), which is located in a deep cleft between the two domains (Fig. 2). The cofactor and the substrate access the catalytic site from opposite sides. While Cys-241 is readily accessible from the cofactor site, substrates access the catalytic site through a deep funnel with an approximate length of 25 Å (Fig. 3). The cofactor-binding site is highly conserved in the superfamily of ALDHs (Supplementary Fig. 2). In the present structure, residues Ile-108, Trp-111, Asn-112, Glu-138, Ser-140, Val-167, Thr-170, Leu-174, Gly-185, Val-189, Ile-192, Val-193, Trp-231 and Phe-333 are expected to be involved in either hydrogen bonds or van der Waals contacts with the NAD cofactor. All of these residues are conserved between rat ALDH3a1 (1ad3 (ref. 14)) and FALDH.


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)

An additional gatekeeper helix constrains substrate specificity by restricting the substrate binding funnel.(a) Cross section through the active site of FALDH. The NAD-binding pocket and the substrate funnel positioned on opposite sides of the enzyme and merge in its central active site. Active site residues are indicated in red (Asn-112, Glu-207, Cys-241 and Glu-331). The electron density found in the substrate funnel is indicated in yellow. The electron density mesh was generated at 1.2 sigma within 1.6 Å around a hexadecanoic acid molecule, which was previously modelled into the electron density. The gatekeeper helix, covering the substrate funnel entrance is shown in green. The substrate funnels of (b) FALDH and (c) ALDH3A1 (3sza) were characterized using the MOLE toolkit for PyMOL. While in FALDH, the additional gatekeeper helix (shown in green) creates a kink in the channel (shown in yellow), the wide, linear arrangement of the substrate funnel entrance of ALDH3A1 is not restricted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: An additional gatekeeper helix constrains substrate specificity by restricting the substrate binding funnel.(a) Cross section through the active site of FALDH. The NAD-binding pocket and the substrate funnel positioned on opposite sides of the enzyme and merge in its central active site. Active site residues are indicated in red (Asn-112, Glu-207, Cys-241 and Glu-331). The electron density found in the substrate funnel is indicated in yellow. The electron density mesh was generated at 1.2 sigma within 1.6 Å around a hexadecanoic acid molecule, which was previously modelled into the electron density. The gatekeeper helix, covering the substrate funnel entrance is shown in green. The substrate funnels of (b) FALDH and (c) ALDH3A1 (3sza) were characterized using the MOLE toolkit for PyMOL. While in FALDH, the additional gatekeeper helix (shown in green) creates a kink in the channel (shown in yellow), the wide, linear arrangement of the substrate funnel entrance of ALDH3A1 is not restricted.
Mentions: The catalytic and cofactor-binding domains of human FALDH (huFALDH) are structurally similar to that of cytosolic rat and human ALDH3A1 (with r.m.s.d. of 0.718 and 0.543, respectively) and contain all the structural features that characterize the ALDH family141620. The catalytic domain contains the catalytic cysteine (Cys-241, corresponding to Cys-302 in mammalian class 1 and class 2 ALDHs16), which is located in a deep cleft between the two domains (Fig. 2). The cofactor and the substrate access the catalytic site from opposite sides. While Cys-241 is readily accessible from the cofactor site, substrates access the catalytic site through a deep funnel with an approximate length of 25 Å (Fig. 3). The cofactor-binding site is highly conserved in the superfamily of ALDHs (Supplementary Fig. 2). In the present structure, residues Ile-108, Trp-111, Asn-112, Glu-138, Ser-140, Val-167, Thr-170, Leu-174, Gly-185, Val-189, Ile-192, Val-193, Trp-231 and Phe-333 are expected to be involved in either hydrogen bonds or van der Waals contacts with the NAD cofactor. All of these residues are conserved between rat ALDH3a1 (1ad3 (ref. 14)) and FALDH.

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