Site‐directed mutagenesis of histidine residues in the Δ12 acyl‐lipid desaturase of <i>Synechocystis</i>

Avelange‐Macherel, Marie‐Hélène; Macherel, David; Wada, Hajime; Murata, Norio

doi:10.1016/0014-5793(95)00163-4

Cited by 65 publications

(60 citation statements)

References 32 publications

(40 reference statements)

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“…First, we have identified the ⌬5-Des essential His clusters involved in iron ligation by site-directed mutagenesis, which are coincident with those already identified in rat ⌬ 9 and cyanobacterial ⌬ 12 desaturases (9,26). Our data also allow us to discard the possibility that the fourth His-rich region, which is found exclusively in the B. subtilis ⌬5-Des, is involved in the active site.…”

Section: Discussionsupporting

confidence: 71%

Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

Díaz¹,

Mansilla²,

Vila³

et al. 2002

Journal of Biological Chemistry

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The Bacillus subtilis acyl-lipid desaturase (⌬5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of ⌬5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of ⌬5-Des with the reporter alkaline phosphatase (⌬5-Des-PhoA). The alkaline phosphatase activities of cells expressing the ⌬5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for ⌬5-Des as a polytopic membrane protein with six transmembrane-and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.The fatty acyl desaturases encompass a family of enzymes, representatives of which are found in all lower eukaryotes, plants and animals (for a review see Ref. 1), and some prokaryotes such as cyanobacteria (2) and bacilli (3), which have the function of introducing double bonds into fatty acyl chains.Although they all utilize molecular oxygen and reducing equivalents obtained from an electron transport chain, and the basic mechanism of the desaturation reaction may be very similar in all cases (4), fatty acid desaturases can be classified into three main subfamilies (1): (i) the soluble acyl carrier protein desaturases that introduce double bonds into fatty acids esterified to acyl carrier protein and are found in the stroma of plant plastids (4); (ii) the acyl-lipid desaturases, which are membranebound enzymes associated with the endoplasmic reticulum (1), the plant chloroplast membrane (5), the cytoplasmic membrane of some bacilli (6), and the plasmatic and thylacoid membranes of cyanobacteria (7) that desaturate fatty acids esterified in glycerolipids; (iii) the acyl-CoA desaturases, which introduce double bonds into fatty acids esterified to CoA (8) and are associated to the endoplasmic reticulum membrane of animals and fungi (1).The soluble and the membrane-bound desaturases show different consensus motifs (for a review, see 4). Database searching for these motifs reveals that they belong to two distinct multifunctional classes of iron-dependent enzymes, each of which includes desaturases, hydroxylases, and epoxidases that act on fatty acids ...

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Section: Discussionsupporting

confidence: 71%

Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

Díaz¹,

Mansilla²,

Vila³

et al. 2002

Journal of Biological Chemistry

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“…Mammalian ELOVL enzymes (ELOVL1-7) catalyze distinct elongation steps, mediating the first and rate-limiting condensation reaction catalyzed by iron-coordinating histidines during fatty acid elongation (40)(41)(42)(43), a process that occurs in the ER. Since mutations in ELOVL4 occur downstream of the active site histidines, the possibility remains that the truncated gene product may be enzymatically active, generating keto intermediates of fatty acid elongation in cellular compartments where it may be toxic.…”

Section: Discussionmentioning

confidence: 99%

Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy

Logan¹,

Agbaga

Chan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Autosomal-dominant Stargardt-like macular dystrophy [Stargardt3 (STGD3)] results from single allelic mutations in the elongation of very-long-chain fatty acids-like 4 (ELOVL4), whereas recessive mutations lead to skin and brain dysfunction. ELOVL4 protein localizes to the endoplasmic reticulum, where it mediates the condensation reaction catalyzing the formation of very-long-chain (VLC) (C-28 to C-40) fatty acids, saturated and polyunsaturated (PUFA). The defective gene product is truncated at the C terminus, leading to mislocalization and aggregation in other organelles. We hypothesized that the STGD3 truncated mutant may generate mislocalized, and therefore toxic, keto intermediates of fatty acid elongation, thereby contributing to the disease process. Using cell-based and cell-free microsome assays, we found that the truncated protein lacked innate condensation activity. Coexpression of different forms of wild-type and mutant ELOVL4 revealed a large dominant-negative effect of mutant protein on ELOVL4 localization and enzymatic activity, resulting in reduced VLC-PUFA synthesis. The reduction in VLC-PUFA levels in STGD3 and age-related macular degeneration may be a contributing factor to their retinal pathology.Stargardt disease | condensing enzyme | photoreceptor degeneration

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“…These conserved histidine residues have been proposed as ligands to a diiron active site cluster identified by Mossbauer spectroscopy in the distantly related alkane -hydroxylase enzyme from Pseudomonas oleovorans (21). Functional relevance of the histidine cluster has been determined for rat and cyanobacterial desaturases and also in the alkane -hydroxylase (22). However, the relevance of these residues has not been described for Ole1p.…”

Section: Co-expression Of Wild Type and Inactive Mutant Desaturase Prmentioning

confidence: 99%

Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

Lou

Shanklin

2010

Journal of Biological Chemistry

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Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the ⌬ 9 -acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an ϳ75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.Unsaturated fatty acids are found in the membranes of prokaryotes and eukaryotes. The balance between saturated and unsaturated fatty acids is a key variable in maintaining membrane fluidity. Postsynthetic desaturation of fatty acids is mediated by desaturase enzymes (1-5). Fatty acid desaturases are derived from two evolutionary lineages, the soluble acyl-ACP desaturases found primarily in the plastids of higher plants (e.g. the castor ⌬ 9 -18:0-acyl 2 carrier protein (ACP) 3 desaturase (6) and the integral membrane desaturases typified by the yeast Ole1 ⌬ 9 -desaturase (Ole1p) (7)). The reactions require iron cofactors, molecular oxygen, and reducing equivalents and are both inhibited by azide and cyanide but are insensitive to carbon monoxide. All desaturases investigated to date show remarkable stereo-selectivity in abstracting pro(R) hydrogens from adjacent carbons in the fatty acid substrate (8). As noted by Bloch (1), desaturase selectivity "would seem to approach the limits of the discriminatory power of enzymes" by a mechanism(s) that remain to be determined almost 40 years later.Ole1p is a bifunctional 53-kDa protein (7), composed of two distinct domains: an N-terminal 42-kDa desaturase domain that has 36% homology to the rat stearoyl-CoA desaturase and a C-terminal 11-kDa domain that has strong homology to cytochromes b 5 (9).Although integral membrane desaturases, like many membrane proteins, have proved difficult overexpress and purify, sequence analysis has provided important information regarding the structure of the enzyme. For example, Martin and coworkers (7) employed hydropathy analysis to predict t...

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Site‐directed mutagenesis of histidine residues in the Δ12 acyl‐lipid desaturase of Synechocystis

Cited by 65 publications

References 32 publications

Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy

Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

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