scholarly journals Consensus mutagenesis and ancestral reconstruction provide insight into the substrate specificity and evolution of the front-end Δ6-desaturase family

2020 ◽  
Author(s):  
Dongdi Li ◽  
Adam M. Damry ◽  
James R. Petrie ◽  
Thomas Vanhercke ◽  
Surinder P. Singh ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Marine Algae ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Ancestral Reconstruction ◽  
Micromonas Pusilla ◽  
Ancestral Protein ◽  
Δ6 Desaturase

ABSTRACTMarine algae are a major source of omega (ω)-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae begins with the desaturation of fatty acids by Δ6-desaturases and enzymes from different species display a range of specificities towards ω3 and ω6 LCPUFAs. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This dataset provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.

scholarly journals Plastidic Δ6 fatty-acid desaturases with distinctive substrate specificity regulate the pool of c18-pufas in the ancestral picoalga ostreococcus tauri

2020 ◽  
Author(s):  
Charlotte Degraeve-Guilbault C. ◽  
Rodrigo E. Gomez ◽  
Cécile. Lemoigne ◽  
Nattiwong Pankansem ◽  
Soizic Morin ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Acyl Chain ◽  
Cytochrome B5 ◽  
Head Group ◽  
Fatty Acid Desaturases ◽  
Physiological Alterations ◽  
Lipid Head Group ◽  
Species Specific ◽  
Native Host ◽  
Δ6 Desaturase

ABSTRACTEukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several microalgae, including O. tauri, plastidic C18-PUFA are specifically regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. Sequence retrieval from O. tauri desaturases, highlighted two putative Δ6/Δ8-desaturases sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 desaturases. Their overexpression in heterologous hosts, including N. benthamiana and Synechocystis, unveiled their Δ6-desaturation activity and plastid localization. O. tauri lines overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount under phosphate starvation but didn’t show any obvious physiological alterations. Detailed lipid analyses from the various overexpressing hosts, unravelled that the substrate features involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native host. The distribution of plastidial Δ6-desaturase products in eukaryotic hosts suggested the occurrence of C18-PUFA export from the plastid.One sentence summaryOsteococcus tauri plastidic lipid C18-PUFA remodelling involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for both head-group and acyl-chain.

scholarly journals Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End Δ6-Desaturase Family

Biochemistry ◽  
2020 ◽  
Vol 59 (14) ◽  
pp. 1398-1409 ◽  
Author(s):  
Dongdi Li ◽  
Adam M. Damry ◽  
James R. Petrie ◽  
Thomas Vanhercke ◽  
Surinder P. Singh ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Ancestral Reconstruction ◽  
Front End ◽  
Insight Into ◽  
Δ6 Desaturase

scholarly journals Biochemical and Structural Characterization of OXA-405, an OXA-48 Variant with Extended-Spectrum β-Lactamase Activity

2019 ◽  
Vol 8 (1) ◽  
pp. 24 ◽  
Author(s):  
Saoussen Oueslati ◽  
Pascal Retailleau ◽  
Ludovic Marchini ◽  
Laurent Dortet ◽  
Rémy A. Bonnin ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Structural Changes ◽  
3D Structure ◽  
Hydrolytic Activity ◽  
Structural Basis ◽  
Class D ◽  
Covalent Docking ◽  
Dynamics Simulations ◽  
Hydrolysis Of

OXA-48-producing Enterobacterales have now widely disseminated globally. A sign of their extensive spread is the identification of an increasing number of OXA-48 variants. Among them, three are particularly interesting, OXA-163, OXA-247 and OXA-405, since they have lost carbapenem activities and gained expanded-spectrum cephalosporin hydrolytic activity subsequent to a four amino-acid (AA) deletion in the β5–β6 loop. We investigated the mechanisms responsible for substrate specificity of OXA-405. Kinetic parameters confirmed that OXA-405 has a hydrolytic profile compatible with an ESBL (hydrolysis of expanded spectrum cephalosporins and susceptibility to class A inhibitors). Molecular modeling techniques and 3D structure determination show that the overall dimeric structure of OXA-405 is very similar to that of OXA-48, except for the β5–β6 loop, which is shorter for OXA-405, suggesting that the length of the β5–β6 loop is critical for substrate specificity. Covalent docking with selected substrates and molecular dynamics simulations evidenced the structural changes induced by substrate binding, as well as the distribution of water molecules in the active site and their role in substrate hydrolysis. All this data may represent the structural basis for the design of new and efficient class D inhibitors.

scholarly journals Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity.

1987 ◽  
Vol 262 (8) ◽  
pp. 3754-3761
Author(s):  
A.J. Ganzhorn ◽  
D.W. Green ◽  
A.D. Hershey ◽  
R.M. Gould ◽  
B.V. Plapp
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue ◽  
Kinetic Characterization ◽  
Alcohol Dehydrogenases

Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis

2021 ◽  
Author(s):  
Ryushi Kawakami ◽  
Chinatsu Kinoshita ◽  
Tomoki Kawase ◽  
Mikio Sato ◽  
Junji Hayashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Enzyme Stability ◽  
Hyperthermophilic Archaea ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
Aminobutyrate Aminotransferase ◽  
Chaperone Protein ◽  
Amino Acid Racemase

Abstract The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by co-expression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and non-substrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

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