Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains

<p>Abstract</p> <p>Background</p> <p>Activation of fatty acids by acyl-CoA synthetase enzymes is required for <it>de novo </it>lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6.</p> <p>Results</p> <p>Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms.</p> <p>Conclusion</p> <p>The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.</p

Breaking down barriers: construction of a hybrid heterochiral membrane

Because of a chemical disparity between Archaeal and Bacterial membrane-lipids, these organisms thrive under distinct environmental conditions. Archaea are generally more resistant to extreme habitats like low pH, high temperature or presence of solvents. It has therefore long been hypothesized that the archaeal lipids provide archaeal cells with a higher robustness than bacterial lipids do for Bacteria. A recent study in which bacterial and archaeal lipids were mixed to form hybrid vesicles “lipid enclosed round structures”, for instance showed a higher temperature dependent stability than either the bacterial or archaeal lipid vesicles separately. In the present study, we therefore introduced the enzymatic machinery for assembly of archaeal lipids into the bacterium Escherichia coli. This engineering led to cells with a mixed membrane at a surprisingly high amount of 28% archaeal lipids. Although the intervention led to severe morphological malformations, the cells indeed showed an increased robustness to extreme cold and butanol

Adaptations of archaeal and bacterial membranes to variations in temperature, pH and pressure

The cytoplasmic membrane of a prokaryotic cell consists of a lipid bilayer or a monolayer that shields the cellular content from the environment. In addition, the membrane contains proteins that are responsible for transport of proteins and metabolites as well as for signalling and energy transduction. Maintenance of the functionality of the membrane during changing environmental conditions relies on the cell’s potential to rapidly adjust the lipid composition of its membrane. Despite the fundamental chemical differences between bacterial ester lipids and archaeal ether lipids, both types are functional under a wide range of environmental conditions. We here provide an overview of archaeal and bacterial strategies of changing the lipid compositions of their membranes. Some molecular adjustments are unique for archaea or bacteria, whereas others are shared between the two domains. Strikingly, shared adjustments were predominantly observed near the growth boundaries of bacteria. Here, we demonstrate that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria

Formation of the ether lipids archaetidylglycerol and archaetidylethanolamine in Escherichia coli

In archaea, the membrane phospholipids consist of isoprenoid hydrocarbon chains that are ether-linked to a sn-glycerol 1-phosphate backbone. This unique structure is believed to be vital for the adaptation of these micro-organisms to extreme environments, but it also reflects an evolutionary marker that distinguishes archaea from bacteria and eukaryotes. CDP-archaeol is the central precursor for polar head group attachment. We examined various bacterial enzymes involved in the attachment of L-serine and glycerol as polar head groups for their promiscuity in recognizing CDP-archaeol as a substrate. Using a combination of mutated bacterial and archaeal enzymes, archaetidylethanolamine (AE) and archaetidylglycerol (AG) could be produced in vitro using nine purified enzymes while starting from simple building blocks. The ether lipid pathway constituted by a set of archaeal and bacterial enzymes was introduced into Escherichia coli, which resulted in the biosynthesis of AE and AG. This is a further step in the reprogramming of E. coli for ether lipid biosynthesis.</p

Improving heterologous membrane protein production in Escherichia coli by combining transcriptional tuning and codon usage algorithms.

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overex- pression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting dif- ferent codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool

Improving heterologous membrane protein production in Escherichia coli by combining transcriptional tuning and codon usage algorithms

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overexpression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting different codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool

Disentangling the lipid divide : Identification of key enzymes for the biosynthesis of membrane-spanning and ether lipids in Bacteria

Bacterial membranes are composed of fatty acids (FAs) ester-linked to glycerol-3-phosphate, while archaea have membranes made of isoprenoid chains ether-linked to glycerol-1-phosphate. Many archaeal species organize their membrane as a monolayer of membrane-spanning lipids (MSLs). Exceptions to this "lipid divide"are the production by some bacterial species of (ether-bound) MSLs, formed by tail-to-tail condensation of FAs resulting in the formation of (iso) diabolic acids (DAs), which are the likely precursors of paleoclimatological relevant branched glycerol dialkyl glycerol tetraether molecules. However, the enzymes responsible for their production are unknown. Here, we report the discovery of bacterial enzymes responsible for the condensation reaction of FAs and for ether bond formation and confirm that the building blocks of iso-DA are branched iso-FAs. Phylogenomic analyses of the key biosynthetic genes reveal a much wider diversity of potential MSL (ether)-producing bacteria than previously thought, with importantt implications for our understanding of the evolution of lipid membranes

Study to Estimate the Amount of Disaster Waste Using Remote Sensing Data

博士（工学）秋田大

Transmembrane helix prediction and codon usage landscapes for the different variants for DGGGPs.

<p>(a) Transmembrane helix prediction plot depicting the probability of residues being in a transmembrane helix domain (red bars), on the inside or cytosolic side of the membrane (blue line) or outside of the membrane (purple line) ((TMHMM v2.0). Codon usage landscapes are depicted based on Relative Codon Adaptiveness (RCA) scores for individual amino-acids and a moving average over 5 codons (black line), for (b) the wild-type gene for native host codon usage (<i>M</i>. <i>maripaludis</i> C5); (c) the codon-harmonized gene variant for <i>E</i>. <i>coli</i> codon usage; (d) the codon-optimized gene variant for <i>E</i>. <i>coli</i> codon usage (e) the wild-type gene variant for <i>E</i>. <i>coli</i> codon usage.</p

Membrane-integrated production levels for all codon usage variants.

<p>Production levels in <i>E</i>. <i>coli</i> LEMO(DE3) were determined by whole-cell GFP-fluorescence at different transcriptional tuning by varying the L-rhamnose concentration (indicated in μM). All expression experiments were at least performed in biological triplicates.</p