Molecular cloning and 3D model of a fatty-acid elongase in a carnivorous freshwater teleost, the European perch (Perca fluviatilis)

Abstract

The European perch (Perca fluviatilis) is a carnivorous freshwater fish able to metabolise polyunsaturated fatty acids (PUFA) into highly unsaturated fatty acids (HUFA). This makes it a potential candidate for sustainable aquaculture development. In this study, special attention is given to the fatty-acid elongase (ELOVL) family, one of the two enzymatic systems implied in the HUFA biosynthesis. Structural information on European perch enzyme converting PUFA into HUFA is obtained by both molecular cloning and in silico characterization of an ELOVL5-like elongase from P. fluviatilis (pfELOVL). The full-length cDNA sequence consists of a 885-base pair Open Reading Frame coding for a 294-amino acid protein. Phylogenetic analysis and sequence alignment with fish elongases predict the pfELOVL clusters within the ELOVL5 subgroup. The amino-acid sequence displays the typical ELOVL features: several transmembrane α helices (TMH), an endoplasmic reticulum (ER) retention signal, and four "conserved boxes" involved in the catalytic site. In addition, the topology analysis predicts a 7-TMH structure addressed in the ER membrane. A 3D model of the protein embedded in an ER-like membrane environment is also provided using de novo modelling and molecular dynamics. From docking studies, two putative enzyme-substrate-binding modes, including H bonds and CH-π interactions, emphasize the role of specific residues in the "conserved boxes".

Genome-wide identification and expression profile of Elovl genes in threadfin fish Eleutheronema

“…The secondary structures of these proteins in E. tetradactylum revealed 40.86-50.30% alpha helixes, 28.10-28.10% random coil, 13.75-20.67% extended strand and 2.38-4.47% beta turn, while these ratios were predicted to be 47.55-53.27, 30.00-36.01, 6.99-18.12 and 2.38-4.75%, respectively, in E. rhadinum (Table 3). High ratio of alpha helixes and random coil in the Elovl protein structure might play important roles in fatty acids biosynthesis in fish, in accordance with the literature for the order Perciformes in Perca fluviatilis 36 . Additionally, the secondary structure pattern of Elovl proteins in the candidate E. tetradactylum and E. rhadinum species were highly similar (Fig.…”

Characterization of Three Types of Elongases from Different Fungi and Site-Directed Mutagenesis

“…INVSc1 containing pYES2/NT C-ELOs was incubated on SC-U for 48 h at 28 • C, diluted to an OD600 of 0.4, and then an equal amount was added to the induction medium. Cultivation conditions were maintained at 28 • C for 48 h. When needed, 100 µM fatty acid substrates (NU-CHEK, Elysian, MN, USA), including linoleic acid (LA, C18:2 ∆9,12 ), α-linolenic acid (ALA, C18:3 ∆9,12,15 ), γ-linolenic acid (GLA, C18:3 ∆6,9,12 ), arachidonic acid (ARA, 20:4 ∆5, 8,11,14 ), eicosapentaenoic acid (EPA, 20:5 ∆5,8,11,14,17 ), methyl arachidate (mC20:0), and 0.4% Tergitol NP-40 (Sigma, St. Louis, MO, USA, v/v) for fatty acid solubilization were added to the SC-U induction medium. The cultures were collected and the pellets were used for further analysis.…”

“…Fatty acid analysis of pY-PsELO revealed decreased C16:0 from 20.5% to 15.5%, and new peaks for C20:0 with 2.4% and C22:0 with 0.7% compared to control, suggesting PsELO can sequentially elongate C16:0 to longer chain fatty acids. PY-McELO fatty acid data showed that McELO could convert 8.4% of LA to C20:2 ∆11,14 and 3.2% of ALA to C20:3 ∆11, 14,17 . Quantification results suggested that PrELO elongated 78.…”

“…The substrate specificity-determining sites within the elongase catalytic domain remain experimentally unvalidated, hindering further enzyme modification and application. Recent studies have utilized various methods to investigate functional regions of elongases, including sequence comparison to identify conserved amino acid sites and subsequent individual mutations to pinpoint key catalytic residues [13], peptide chain region exchange to determine enzymatic specificity [9], and molecular simulation to predict functional regions of elongases and their influence on substrate selectivity [14]. Variations in specific amino acid residues among different elongase types may dictate their unique substrate preferences.…”

The Promoter SNPs Were Associated with Both the Contents of Poly-Unsaturated Fatty Acids (PUFAs) and the Expressions of PUFA-Related Genes in Common Carp

“…In fish, fads2 was responsible for introducing double bonds at specific positions of the fatty acyl chain [ 12 ]. The elovl gene family in fish includes multiple elovl genes such as elovl2 , elovl4 , and elovl5 , which have specific substrates in the elongation of PUFAs [ 13 ]. The SNPs in the coding and promoter regions of fads2 and elovl5 have been proven to be associated with the PUFAs content in human [ 14 ], beef cattle [ 15 ], bovine milk [ 16 , 17 ], pigs [ 18 ], and gilthead sea bream [ 19 ].…”