Studies On The Expression, Purification And Substrate Specificities Of Mortierella Alpina Integral Membrane Fatty Acid Desaturases

Polyunsaturated fatty acids?PUFAs?play important roles in human health.They have beneficial effects in the prevention of cancer and cardiovascular disease.?9 desaturase?FADS9?introduce the first double bond into the long chain of fatty acids,?12 desaturase?FADS12?and?15 desaturase?FADS15?are key enzymes in the desaturation pathway.The regio-and stereo-selective introduction of an unsaturated bond by these enzymes is remarkable,given the lack of unique structural or chemical features along the aliphatic chain.However,the genes for the FADS12 and FADS15 have been lost in mammals during evolution.Thus,our studies focused on the FADS9,FADS12 and FADS15 from the oleaginous fungus Mortierella alpina,a model system to produce PUFAs.Due to low expression level and technical difficulties involved in obtaining large quantities of purified membrane bound proteins,current research progress are mostly restricted in gene cloning,expression and in vivo characterization,no detailed biochemical analysis was reported.The study of M.alpina FADS9,FADS12 and FADS15 will not only facilitate the study of fatty acid desaturation mechanism and structure function relationship,but also be invaluable to the production of important PUFAs.This study firstly heterologously expressed and purified the M.alpina FADS9,FADS12 and FADS15,obtained sufficient amounts of pure active proteins.Then,we expressed and purified the electron transfer proteins cytochrome b₅,NADH-cytochrome b₅ reductase and NADPH-cytochrome P450 reductase,constructed the in vitro fatty acid desaturase reaction system.Substrate specificities and catalytic properties of the desaturases were determined.Topology models and three dimentional structures of FADS12 and FADS15 were predicted for the preliminary analysis of the structure and function relationship.Main results are concluded as follows:1.Expression and purification of FADS9,FADS12 and FADS15:The desaturase genes were appended to a cassette containing the human rhinovirus 3C protease cleavage site,the IgG-specific ZZ-tag,and an RGS-His10-tag.The plasmid was transformed into Pichia pastoris and the highest expressing clone was determined.Membrane fraction was isolated by gradient centrifugation and Fos-Choline-16 was selected as the best detergent for efficient extraction of desaturase.IgG affinity purification and cation exchange chromatography of desaturase resulted in homogenous,pure protein.The purified FADS9 was intact and contained fused cytochrome b₅ domain.The purified FADS12 and FADS15 contained a di-iron center.2.Construction of the in vitro fatty acid desaturase reaction system:The Cytb5,Cytb5R and CytP450R gene were appended to a His6-tag and transformed into Escherichia coli for expression.The purification using Cobalt affinity,ion exchange and gel filtration resulted in pure active protein.The Cytb5R activity determined using DCIP as substrate was 1072.0 U,and Cytb5R and CytP450R were able to reduce Cytb5 with the presence of NADH and NADPH respectively.3.Substrate specificities and kinetic studies of FADS9,FADS12 and FADS15:Substrate specificities and kinetic parameters of FADS12 and FADS15 were determined by coupling with the Cytb5 and Cytb5R.Results showed that 18:1??9?is the preferred substrate of FADS12,the K_m and k_cat value were 5.4±0.8?M and 0.9±0.04 min^-1 respectively.18:2??6?is the preferred substrate of FADS15,the K_m and k_cat value were 15.9±2.2?M and 11.2±0.6min^-1 respectively.The activity determined using NADPH as reductant was 40⁶0%of using NADH as reductant,the desaturation efficiency was higher in using NADH as reductant.In terms of the FADS9 that contained the fused Cytb5 domain,activity and substrate specificity was determined using yeast lysate system,results showed that FADS9 has similar conversion rate toward 18:0 and 16:0 substrates.4.Preliminary analysis of the structure and function relationship of FADS12 and FADS15.Primary sequence alignments,phylogenetic analysis,PolyPhobius topology prediction and three dimentional structure prediction results revealed that FADS12 and FADS15 have similar overall architecture as FADS9,the transmembrane regions contain four transmembrane?-helices,but the structure of cap regions are significantly different,they contain only two amphipathic?-helix,FADS12 contains a?-sheet,the location of the active site and the substrate binding channel change significantly,these lay the structure foundations for the substrate selectivity of FADS12 and FADS15 and their desaturating functions at different positions.