| The ?-3 fatty acid desaturase(?3Des)is a key enzyme in the biosynthesis of long-chain polyunsaturated fatty acids(LC-PUFAs).It can catalyze a wide range of ?-6 long-chain fatty acids ranging from C16 to C20 and has broad substrate specificities and convert ?-6-polyunsaturates to their ?-3-counterparts.In the past decades,a number of publications reported ?3Des can produce LC-PUFAs in organisms,such as yeasts,microalgae and fungi.However,previous reports have suggested that ?3Des from different species show a profound diversity of substrate specificities towards different fatty acids substrates.In some oleaginous microorganisms,the composition and ratio of ?-6 and ?-3 fatty acids are significantly different.The distribution and content of ?-6 and ?-3 fatty acids in organisms are directly determined by substrate specificity of their inherent ?3Des.Compared to the ?9 fatty acid desaturase(?9Des),which has been full characterized and crystallized,the molecular mechanism of ?3Des in regulating their substrate specificities is still unknown.To understand the LC-PUFAs production and the differences in substrate specificities among ?-3 fatty acid desaturases,the genes of four candidate fatty acid desaturase were first screened out by various bioinformatics tools in this study,and their biological evolutions,transmembrane and topological structures were also analyzed.Then based on the gene of ?3Des from Mortierella alpina(M.alpina)and Rhizophagus irregularis(R.irregularis),the function of ?-3 fatty acid desaturases were studied,and the mechanism of their substrate specificities was confirmed.The key domains regulating the substrate specificity were also determined by a domain swapping method,which 12 recombinant chimeric enzymes were fused and analyzed.At last the distribution of conservative amino acids within and outside the critical domain of ?3Des was compared and analyzed by bioinformatics.A series of mutants were constructed by local site-directed mutagenesis,and their functional validations were completed in Saccharomyces cerevisiae(S.cerevisiae)expression system.And the key sites affecting substrate specificity of ?3Des within the domain were analyzed in details by homology modeling and molecular docking technology.The molecular mechanism of substrate specificity was further elucidated and analyzed to illustrate the reason for different LC-PUFAs production abilities.The main results of this study are as follows:1.After culturing with ?-6 fatty acid substrates in the media,fatty acid profiles of both supernatants and pellets from S.cerevisiae were assessed to study substrate specificities of ?3Des,and two new ?3Des were screened and obtained.Based on the characteristics of conservative sequences of ?3Des,the genes of four candidate fatty acid desaturase oAcFADS17,oBgFADS17,oObFADS17 and oRiFADS17 were selected by using the gene of oPaFADS17 from Pythium aphanidermatum(P.aphanidermatum)as a template.Their expression vectors in S.cerevisiae were constructed and four proteins were successfully expressed after induction.Their activities and substrate specificities were also determined by assessing fatty acid profiles of both supernatants and pellets.The content of ?-3 fatty acid increased significantly and the conversion rates towards ?-6 fatty acid substrates DGLA and AA reached 61.8% and 58.5%,respectively,and the comprehensive catalytic capacity was significantly superior to the reported 17 Des.In this study,fatty acid desaturase(oObFADS17)was found to be existed in marine mollusks for the first time.2.Based on domain swapping,12 recombinant chimeric enzymes were analyzed,and the amino acid sequences of ?3Des between His boxes I and II was the key domain affecting substrate specificity.Considering the similarity and homology of amino acid sequences,we chose the oRiFADS17 gene from R.irregularis and the FADS15 gene from M.alpina as two parent proteins.Both amino acid sequences of two parent proteins were divided into six fragments and replased each other by domain swapping technology.12 genes of recombinant ?3Des(Chimera 1-12)were fused by overlapping extension PCR,and their activities were determined after successfully expressed in S.cerevisiae.The results showed that the ratio of conversion rates towards ?-6 fatty acid substrates in Chimera 3 and Chimera 9 changed significantly,which proved that the amino acid sequences between His boxes I and II played a pivotal role in substrate specificity of ?3Des.3.Through the analysis of amino acid sites within the key domain,we found that W129 and T144 amino acid sites had important effects on the regulation of substrate specificity of ?3Des.Based on multiple sequence alignments and the topological structure information of ?3Des,36 amino acid residues between the sequences of FADS15 from M.alpina and oRiFADS17 from R.irregularis were analyzed for their conservativity.Six completely conserved amino acid residues and four relatively conserved amino acid residues were selected for further study by site-directed mutagenesis.The result indicated that the S118,N124 and G128 amino acid sites had important effects on the catalytic activity of ?3Des,the V137 and V152 amino acid sites played an important role in substrate recognition,and the W129 and T144 amino acid sites played a key role in substrate specificity.At the same time,to figure out which amino acid was the key site that regulating substrate specificity of ?3Des,these sites were analyzed by homology modeling and molecular docking with substrates.The possible reason for the change of substrate specificity was that the mutation of W129 directly acted on the fatty acid part of the substrate,reduced the steric hindrance of entering the active center(contrary to T144),created more space and made the longer chain AA substrates more efficient in entering into or releasing from the substrate channel,thus led to the change of substrate specificity of ?3Des.4.Through the analysis of amino acid sites outside the key domain,we found that A44,M156 and W291 amino acid sites had important effects on the regulation of substrate specificity of ?3Des.FADS15 from M.alpina and oPaFADS17 from P.aphanidermatum were chosen as two parent proteins.Eight relatively conservative amino acid sites were selected for site-directed mutagenesis based on the conservative analysis of their full sequence amino acids by means of multiple sequence alignment and analysis of topological structure.Three key amino acid sites A44,M156 and W291 were determined for regulation of substrate specificity.Cumulative mutants A44S/M156 I,A44S/W291 M,M156I/W291 M and A44S/M156I/W291 M were also constructed and the results showed that there was no synergistic effect among these amino acids.Then another set of three mutants S21 A,I137M and M236 W were constructed in the corresponding sites of oPaFADS17 gene.The results showed that these three amino acid sites affected the catalytic activity of oPaFADS17.Further analysis by molecular docking showed that A44,M156 and W291 sites were closed to the Coenzyme A(CoA)part of the substrate.We speculated that these three sites acted on the CoA carrier part of the substrate,affected the location of the CoA carrier,changed the angle and depth of the substrate and made the longer-chain substrate more efficient in entering into the catalytic active center.The difference of these amino acid sites was the intrinsic reason for the change of substrate selectivity of ?3Des... |
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