Abstract
Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Delta12 desaturase and a processive bifunctional oleoyl-Delta12/linoleoyl-omega3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Delta12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Delta12/omega3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.
Highlights
Metabolic processes are often controlled by the presence of highly specific enzymes
The strict substrate- and regiospecificities are of physiological relevance, because the major mechanism controlling the biophysical properties of a membrane, aside from changing its overall lipid class composition, is the modification of acyl chain length, position, and number of the double bonds in glycerolipids [3]
The resulting organic phase was dried under nitrogen stream and dissolved in 800 l of methanol. 400 l of each sample was transesterified with sodium methoxide to convert lysophospholipids to Fatty acid methyl esters (FAMEs). 6.5 l of trimethylsilyldiazomethane was added to the rest of each sample to convert free fatty acids into their corresponding methyl esters, shaken for 30 min at room temperature, and 0.2 l of acetic acid was added to degrade remaining trimethylsilyldiazomethane
Summary
Materials—Restriction enzymes and DNA-modifying enzymes were obtained from MBI Fermentas. The ORFs were cloned into the amplification plasmid pGEM-T (Promega) and moved as EcoRI/ XhoI fragments into the yeast expression vector pYES2/CT (Invitrogen) in-frame with the sequence encoding a C-terminal V5-tag, yielding pYES2/CT-An2 and pYES2/CT-An1. The hybrid genes were cloned into the amplification plasmid pGEM-T (Promega) and moved as EcoRI/XhoI fragments into the yeast expression vector pYES2/CT (Invitrogen) in-frame with the sequence encoding a C-terminal V5 tag. Lipid analysis of transgenic yeast cells was performed after harvesting and lyophilization of 100-ml cultures supplemented with linoleic acid or without additional fatty acids. 400 l of each sample was transesterified with sodium methoxide to convert lysophospholipids to FAMEs. 6.5 l of trimethylsilyldiazomethane was added to the rest of each sample to convert free fatty acids into their corresponding methyl esters, shaken for 30 min at room temperature, and 0.2 l of acetic acid was added to degrade remaining trimethylsilyldiazomethane. The detector was set to an electron energy of 70 eV, an ion source temperature of 230 °C, and a transfer line temperature of 260 °C
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