Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

Sasata, Robert J.; Reed, Darwin W.; Loewen, Michèle C.; Covello, Patrick S.

doi:10.1074/jbc.m405712200

Cited by 15 publications

(13 citation statements)

References 32 publications

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“…Based on amino acid sequences and catalytic characteristics, the membrane-bound ␤-carotene ketolases belong to a superfamily of integral membrane proteins that catalyze oxidative reactions. This group of proteins share the putative iron-binding His motifs (6,24,27) and have transmembrane domains. Figure 2 shows the similarities in amino acid sequences for a group of CrtW ketolases.…”

Section: Resultsmentioning

confidence: 99%

“…Based on structural and catalytic similarities, the ␤-carotene ketolase encoded by crtW gene belongs to the superfamily of integral membrane oxidative enzymes along with fatty acid desaturases, alkane hydroxylase, xylene monooxygenase, sterol methyloxidase, and ␤-carotene hydroxylase (4,6,24,26,27). Although there is a lack of three-dimensional structural information for these membrane proteins, the easily noticeable features include the three histidine-rich motifs for iron coordination and hydrophobicity patterns.…”

Section: Discussionmentioning

confidence: 99%

“…It has been proposed that the hydrophobic segment between the first two His clusters in the Bacillus subtilis acyl-lipid desaturase may correspond to a protein region that is involved in substrate interaction (6). In the fatty acid desaturase of Caenorhabditis elegans, the C-terminal domain and one of the hydrophobic segments determine the regioselectivity (24). In the CrtW proteins, there are at least two hydrophobic patches, one located between the first two His clusters and the other near the C terminus (Fig.…”

Section: Vol 72 2006 Analysis Of ␤-Carotene Ketolase 5835mentioning

confidence: 99%

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Mutational and Functional Analysis of the β-Carotene Ketolase Involved in the Production of Canthaxanthin and Astaxanthin

Stead

Yao

et al. 2006

Appl Environ Microbiol

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Biosynthesis of the commercial carotenoids canthaxanthin and astaxanthin requires ␤-carotene ketolase. The functional importance of the conserved amino acid residues of this enzyme from Paracoccus sp. strain N81106 (formerly classified as Agrobacterium aurantiacum) was analyzed by alanine-scanning mutagenesis. Mutations in the three highly conserved histidine motifs involved in iron coordination abolished its ability to catalyze the formation of ketocarotenoids. This supports the hypothesis that the CrtW ketolase belongs to the family of iron-dependent integral membrane proteins. Most of the mutations generated at other highly conserved residues resulted in partial activity. All partially active mutants showed a higher amount of adonixanthin accumulation than did the wild type when expressed in Escherichia coli cells harboring the zeaxanthin biosynthetic gene cluster. Some of the partially active mutants also produced a significant amount of echinenone when expressed in cells producing ␤-carotene. In fact, expression of a mutant carrying D117A resulted in the accumulation of echinenone as the predominant carotenoid. These observations indicate that partial inactivation of the CrtW ketolase can often lead to the production of monoketolated intermediates. In order to improve the conversion rate of astaxanthin catalyzed by the CrtW ketolase, a color screening system was developed. Three randomly generated mutants, carrying L175M, M99V, and M99I, were identified to have improved activity. These mutants are potentially useful in pathway engineering for the production of astaxanthin.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Vol 72 2006 Analysis Of ␤-Carotene Ketolase 5835mentioning

confidence: 99%

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Mutational and Functional Analysis of the β-Carotene Ketolase Involved in the Production of Canthaxanthin and Astaxanthin

Stead

Yao

et al. 2006

Appl Environ Microbiol

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“…Provided the Gnathostomata (jawed fi sh) ancestral Fads2 had ⌬ 6 desaturase activity ( 32 ), as for mammalian FADS2s ( 35 ), the expansion of Fads2s in teleosts has been accompanied by subfunctionalization in the enzyme derived from independent mutations in the primary aa sequence ( 32 ). Although a recent study identifi ed a single aa residue as determining the differential ability for 22:5n-3 elongation between ELOVL2 and ELOVL5 elongases in rat ( 48 ), identifi cation of specifi c domains/residues controlling the functionality of desaturases has been elusive and studies are restricted to nonvertebrate enzymes (49)(50)(51). However, the above said, the existence of three ⌬ 6 ⌬ 5 desaturases and three ⌬ 4 desaturases distributed in four distinct species, allows us to explore potential evolutionary scenarios for teleostei Fads2 subfunctionalization.…”

Section: Discussionmentioning

confidence: 99%

Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor

Fonseca-Madrigal

Navarro

Hontoria

et al. 2014

Journal of Lipid Research

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Biosynthesis of long-chain PUFAs (LC-PUFAs) in vertebrates involves sequential desaturation and elongation of C 18 PUFA, linoleic acid (LOA; 18:2n-6), and ␣ -linolenic acid (LNA; 18:3n-3) ( 1 ). Synthesis of arachidonic acid (ARA; 20:4n-6) and EPA (20:5n-3) from LOA and LNA, respectively, utilizes the same enzymes and pathways. The pre dominant pathway involves ⌬ 6 desaturation of LOA or LNA to 18:3n-6/18:4n-3 that are elongated to 20:3n-6/20:4n-3 followed by ⌬ 5 desaturation to ARA/EPA ( 1 ), but an alternative pathway with initial elongation of LOA or LNA followed by ⌬ 8 desaturation, an inherent ability of some ⌬ 6 desaturases, may be possible ( 2 ). Biosynthesis of DHA (22:6n-3) from EPA can also occur by two pathways. First, the so-called "Sprecher pathway" involves two sequential elongation steps from EPA to 24:5n-3 and a subsequent ⌬ 6 desaturation to 24:6n-3, followed by peroxisomal chain shortening ( 3 ). Second, a more direct pathway has been postulated in some marine fi sh that involves elongation of EPA to docosapentaenoic acid (22:5n-3) followed by ⌬ 4 desaturation to DHA ( 4,5 ).Dietary PUFAs are essential in fi sh, although requirements vary with species ( 6, 7 ). Generally, C 18 PUFAs can satisfy essential FA requirements of freshwater and salmonid species, but most marine fi sh have a requirement for LC-PUFAs such as EPA and DHA ( 8 ). Differing essential FA requirements have been linked to differences in the complement of fatty acyl desaturase (Fads) and elongase of very longchain FA (Elovl) genes ( 9-31 ). Thus, the dependence of Abstract Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fi sh is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor , one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C 18 PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying ⌬ 4 and ⌬ 6/ ⌬ 5 specifi cities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C 18 PUFA. In addition, the specifi cities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fi sh lineages. -GA-2010-276916, LONGFA). Additional funding was obtained from CONACYT, Mexico (INSAM FOINS 102/201...

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“…This includes the study of their role in stress response mechanisms [21,22]. Moreover, the study by Sasata et al [23] on regioselectivity of a membranebound desaturase also showed important roles for the Nand C-termini of this enzyme, which are located in the cytoplasmic phase. The present work began with D 6 desaturase, the only desaturase which has shown responsiveness to low temperature [24].…”

Section: Resultsmentioning

confidence: 99%

Truncation Mutants Highlight a Critical Role for the N- and C-termini of the Spirulina Δ6 Desaturase in Determining Regioselectivity

et al. 2007

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We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membraneassociated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.

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Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

Cited by 15 publications

References 32 publications

Mutational and Functional Analysis of the β-Carotene Ketolase Involved in the Production of Canthaxanthin and Astaxanthin

Mutational and Functional Analysis of the β-Carotene Ketolase Involved in the Production of Canthaxanthin and Astaxanthin

Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor

Truncation Mutants Highlight a Critical Role for the N- and C-termini of the Spirulina Δ6 Desaturase in Determining Regioselectivity

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