Molecular Cloning and Functional Expression of a Δ9- Fatty Acid Desaturase from an Antarctic Pseudomonas sp. A3

Garba, Lawal; Ali, Mohd Shukuri Mohamad; Oslan, Siti Nurbaya; Rahman, Raja Noor Zaliha Raja Abdul

doi:10.1371/journal.pone.0160681

Cited by 15 publications

(13 citation statements)

References 35 publications

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“…A3 and cold‐tolerant Pseudomonas sp. A8, the genes PA3FAD9 and PA8FAD9 coding, respectively, for a membrane Δ9‐FA desaturase protein . Similar to detected in our study with the expression of desA of P. putida A (ATCC 12633), the functional expression of the genes PA3FAD9 and PA8FAD9 in E. coli transetta (DE3) displayed a significant increase in the proportions of palmitoleic acid content in recombinant E. coli cells, confirmimg an active Δ9‐FA protein.…”

Section: Discussionsupporting

confidence: 87%

“…) that are conserved in most membrane desaturases and that have been shown to be important for catalysis and presumed to compose the Fe‐binding active centers of these enzymes . The Kyte‐Doolittle hydropathy profiles of DesA revealed two hydrophobic regions (between amino acids 10–33 and 137–160) similar to the sequence profiles of known membrane‐associated desaturases . Although this study does not advance on the purification of this enzyme, the characteristics observed in the predicted DesA protein (histidine boxes and hydrophobic domains) are consistent with the topological models derived from studies performed with other desaturases, such as yeast A9 acyl‐CoA desaturase , mouse steaoryl‐CoA desaturase , and acyl lipid Δ5 desaturase from Bacillus subtilis .…”

Section: Discussionsupporting

confidence: 78%

“…A8 (GenBank accession no: AMX81567.1) , P. aeruginosa PAO1(GenBank accession no:NP_248977.1) , P. fluorescens A506 (GenBank accession no: AFJ57994.1) , and Pseudomonas sp. A3 (GenBank accession no: KT033499) . Three conserved histidine boxes are indicated by I, II, and III for HXXXXH, HXXHH, and HXXHH, respectively…”

Section: Resultsmentioning

confidence: 99%

“…In Pseudomonas sp A8 , Pseudomonas sp. A3 , and in Pseudoalteromonas sp. MLY15 , oxygen‐dependent FA desaturation catalyzed by desaturase enzymes has an important role in increasing UFA content in lipids, in such a way that it contributes to membrane fluidity when cells are exposed to low temperature stress.…”

Section: Introductionmentioning

confidence: 99%

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Pseudomonas putidaΔ9‐fatty acid desaturase: Gene cloning, expression, and function in the cationic surfactants stress

Heredia

Lucchesi

2019

J Basic Microbiol

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Pseudomonas putida counteract the fluidizing effect of cationic surfactants decreasing the content of membrane unsaturated fatty acid (UFA). A Δ9-fatty acid desaturase gene (desA) from P. putida was isolated, cloned, and successfully expressed in Escherichia coli, a Δ9 desaturase deficient organism. desA consists of 1185 bp and codes for 394 amino acids. The deduced amino acid sequence reveals three histidine clusters and a hydropathy profile, typical of membrane-bound desaturases. Validating desA expression in E. coli cells, the amount of palmitoleic acid increased from 2.05 to 7.36%, with the concomitant increase in membrane fluidity (fluorescence polarization value decrease from 0.13 ± 0.03 to 0.09 ± 0.02).Also, when DesA activity was assayed in vivo, the percentage of UFA obtained from exogenous palmitic acid [1-14 C] increased 10-fold. In contrast, when cells expressing desA were exposed 15 min at sublethal concentration of cationic surfactants, the amount of UFA was 82% lower than that detected in cells non-exposed. Thus, the decrease in UFA content to counteract the fluidizing effect of cationic surfactants can be correlated with reduction of DesA activity. K E Y W O R D Sdesaturase enzymes, desaturase gene, Pseudomonas putida, quaternary ammonium surfactants, unsaturated fatty acids

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Section: Discussionsupporting

confidence: 87%

Section: Discussionsupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Pseudomonas putidaΔ9‐fatty acid desaturase: Gene cloning, expression, and function in the cationic surfactants stress

Heredia

Lucchesi

2019

J Basic Microbiol

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“…Pseudomonas sp. AMS8 ∆9-fatty acid desaturase and sequences from several other ∆9-fatty acid desaturases has revealed the three conserved-histidine boxes common to all membranebound desaturases in bacteria (Garba et al, 2016b;Li et al, 2009), fungi (Chen et al, 2013) and animals (Bai et al, 2015) (Figure 1). The human integral membrane stearoyl-CoA desaturase (PDB ID: 4ZYO) and mouse stearoyl-coA desaturase (PDB ID: 4YMK) have been solved to a resolution of 3.25 Å and 2.6 Å, respectively.…”

Section: Sequence Of the ∆9-fatty Acid Desaturase Protein And Templatmentioning

confidence: 99%

Peer Review #1 of "Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 (v0.2)"

2018

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Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The ∆9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The Cold-tolerant Pseudomonas sp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active ∆9-fatty acid desaturase was isolated and functionally expressed in Escherichia coli. In this paper we report homology modeling and docking studies of a ∆9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plot and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy,-6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely,

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Effects of a Δ-9-fatty acid desaturase and a cyclopropane-fatty acid synthase from the novel psychrophile Pseudomonas sp. B14-6 on bacterial membrane properties

Choi

Kim

Song

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.

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Molecular Cloning and Functional Expression of a Δ9- Fatty Acid Desaturase from an Antarctic Pseudomonas sp. A3

Cited by 15 publications

References 35 publications

Pseudomonas putidaΔ9‐fatty acid desaturase: Gene cloning, expression, and function in the cationic surfactants stress

Pseudomonas putidaΔ9‐fatty acid desaturase: Gene cloning, expression, and function in the cationic surfactants stress

Peer Review #1 of "Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 (v0.2)"

Effects of a Δ-9-fatty acid desaturase and a cyclopropane-fatty acid synthase from the novel psychrophile Pseudomonas sp. B14-6 on bacterial membrane properties

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