Restoration of acyl-lipid desaturase activity by a single point mutation of the Des2 protein from Bacillus licheniformis

SASTRE, DIEGO; UTTARO, A; DE MENDOZA, D; ALTABE, SILVIA

Rosario

Congreso; IX-SAMIGE; 2013

Sociedad Argentina de Microbiologia General

Fatty acid desaturases are enzymes that introduce double bonds with high stereo and regioselectivity into fatty acyl chains to produce unsaturated fatty acids (UFAs). These enzymes play a key role in the maintenance of the proper structure and functioning of biological membranes. The aim of this study was to elucidate the structural determinants of activity/specificity of the acyl-lipid desaturases from different Bacillus species. In a previous work we characterized the acyl lipid desaturases present in Bacillus cereus ATCC14579 and Bacillus licheniformis ATCC14580. B. cereus contains two well active acyl-lipid desaturases named Des5 and Des10, respectively. On the other hand, B. licheniformis ATCC 14580 contains two ORF encoding for putative desaturases (des1 and des2). Des1 and Des2 share 66% identity, and both enzymes show all characteristic features of membrane-bound desaturases, including three histidine boxes and transmembrane (TM) domains. Functional expression of Des1 and Des2 in B. subtilis des (-) strain indicated that Des1 is a Δ5 desaturase, whereas Des2 was not active; however, when expressed in E. coli, Des2 showed Δ5 activity. In order to establish the molecular bases of these observed differences, we constructed a series of chimeric enzymes by domain swapping between the active Des5 from B. cereus and Des2 desaturases. By heterologous expression in B. subtilis des (-), we determined that the replacement of the first two Des2 TM domains with the corresponding TM from Des5 was sufficient to activate this enzyme. Additionally, to identify the potential key amino acid residues required for the desaturation activity we used site-directed mutagenesis based on multiple sequence alignments of desaturases and using des2 gene as a template. Surprisingly, we were able to restore the desaturase activity performing a single substitution of a critical residue of Cysteine 40 to Tyrosine (C40Y) localized in the first TM domain close to the lipid-water interphase, likely by anti-snorkeling effect. Interestly, we also found that the acyl-lipid desaturase activity and specificity were influenced by the metabolic context because Des2 (C40Y) in E. coli membranes was more active and displayed different substrate specificity. The information gained from this research could potentially lead to the design of desaturases capable of producing industrially useful UFAs.