Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C−C Bond‐Forming Enzyme

Xu, Guangcai; Kunzendorf, Andreas; Crotti, Michele; Rozeboom, H.J.; Thunnissen, A.M.W.H.; Poelarends, Gerrit J.

doi:10.1002/anie.202113970

Cited by 20 publications

(5 citation statements)

References 30 publications

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“…Homologous genes have similar evolutionary histories, indicating that they evolved from a common ancestor ( 87 ). Various sources, such as gene duplication, exon shuffling, and gene fusion, contribute to the creation of new genes ( 88 , 89 ). Conserved genes are crucial in the basic biology of organisms, while non-conserved genes are responsible for their unique traits ( 26 ).…”

Section: Discussionmentioning

confidence: 99%

Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C)

Rao,

Lunde,

Dolan

et al. 2023

Front. Immunol.

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BackgroundBoth bacterial and viral diseases are a major threat to farmed fish. As the antiviral immune mechanisms in lumpfish (Cyclopterus lumpus L.) are poorly understood, lumpfish leukocytes were stimulated with poly(I:C), a synthetic analog of double stranded RNA, which mimic viral infections, and RNA sequencing was performed.MethodsTo address this gap, we stimulated lumpfish leukocytes with poly(I:C) for 6 and 24 hours and did RNA sequencing with three parallels per timepoint. Genome guided mapping was performed to define differentially expressed genes (DEGs).ResultsImmune genes were identified, and transcriptome-wide analyses of early immune responses showed that 376 and 2372 transcripts were significantly differentially expressed 6 and 24 hours post exposure (hpe) to poly(I:C), respectively. The most enriched GO terms when time had been accounted for, were immune system processes (GO:0002376) and immune response (GO:0006955). Analysis of DEGs showed that among the most highly upregulated genes were TLRs and genes belonging to the RIG-I signaling pathway, including LGP2, STING and MX, as well as IRF3 and IL12A. RIG-I was not identified, but in silico analyses showed that genes encoding proteins involved in pathogen recognition, cell signaling, and cytokines of the TLR and RIG-I signaling pathway are mostly conserved in lumpfish when compared to mammals and other teleost species.ConclusionsOur analyses unravel the innate immune pathways playing a major role in antiviral defense in lumpfish. The information gathered can be used in comparative studies and lay the groundwork for future functional analyses of immune and pathogenicity mechanisms. Such knowledge is also necessary for the development of immunoprophylactic measures for lumpfish, which is extensively cultivated for use as cleaner fish in the aquaculture for removal of sea lice from Atlantic salmon (Salmo salar L.).

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Section: Discussionmentioning

confidence: 99%

Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C)

Rao,

Lunde,

Dolan

et al. 2023

Front. Immunol.

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“…The process of gene duplication and fusion is how new proteins (with new activities) are generated from simpler ancestral proteins. Poelarends and co-workers replicated this process in the laboratory to optimize the biocatalytic potential of 4-OT . In the experiment, two 4-OT protomers (in the canonical 4-OT) were fused by a short linker (GGGAG) that connected the C-terminus of one protomer to the N-terminus of a second protomer.…”

Section: Resultsmentioning

confidence: 99%

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases

et al. 2023

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Members of the 4-oxalocrotonate tautomerase (4-OT) subgroup in the tautomerase superfamily (TSF) are constructed from a single β–α–β unit and form homo- or heterohexamers, whereas those of the other four subgroups are composed of two consecutively joined β–α–β units and form trimers. A subset of sequences, double the length of the short 4-OTs, is found in the 4-OT subgroup. These “fused” 4-OTs form a separate subgroup that connects to the short 4-OTs in a sequence similarity network (SSN). The fused gene can be a template for the other four subgroups, resulting in the diversification of activity. Analysis of the SSN shows that multiple nodes in the fused 4-OTs connect to five linker nodes, which in turn connect to the short 4-OTs. Some fused 4-OTs are symmetric trimers and others are asymmetric trimers. The origin of this asymmetry was investigated by subjecting the sequences in three linker nodes and a closely associated fourth node to kinetic, mutagenic, and structural analyses. The results show that each sequence corresponds to the α- or β-subunit of a heterohexamer that functions as a 4-OT. Mutagenesis indicates that the key residues in both are αPro1 and βArg-11, like that of a typical 4-OT. Crystallographic analysis shows that both heterohexamers are asymmetric, where one heterodimer is flipped 180° relative to the other two heterodimers. The fusion of two subunits (α and β) of one asymmetric heterohexamer generates an asymmetric trimer with 4-OT activity. Hence, asymmetry can be introduced at the heterohexamer level and then retained in the fused trimers.

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“…[ 19 , 20 ] Pleasingly, the here presented 4‐OT catalyzed Michael reactions with 1 as a nucleophile readily proceed under mild conditions in an environmentally friendly aqueous buffer system with ethanol as a green and sustainable cosolvent. [21] In contrast to aromatic nitroalkenes,[ 22 , 23 ] aliphatic nitroalkenes are prone to rapid non‐enzymatic hydration. To outcompete this undesired hydration of the nitroalkenes 2 a – h , we kept reaction times short by using a rather high catalyst loading as well as an excess of acetaldehyde 1 .…”

Section: Discussionmentioning

confidence: 99%

Enantiocomplementary Michael Additions of Acetaldehyde to Aliphatic Nitroalkenes Catalyzed by Proline‐Based Carboligases

2022

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The blockbuster drug Pregabalin is widely prescribed for the treatment of painful diabetic neuropathy. Given the continuous epidemic growth of diabetes, the development of sustainable synthesis routes for Pregabalin and structurally related pharmaceutically active γ-aminobutyric acid (GABA) derivatives is of high interest. Enantioenriched γ-nitroaldehydes are versatile synthons for the production of GABA derivatives, which can be prepared through a Michael-type addition of acetaldehyde to α,β-unsaturated nitroalkenes. Here we report that tailored variants of the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) can accept diverse aliphatic α,β-unsaturated nitroalkenes as substrates for acetaldehyde addition. Highly enantioenriched aliphatic (R)-and (S)-γ-nitroaldehydes were obtained in good yields using two enantiocomplementary 4-OT variants. Our results underscore the synthetic potential of 4-OT for the preparation of structurally diverse synthons for bioactive analogues of Pregabalin.

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Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C−C Bond‐Forming Enzyme

Cited by 20 publications

References 30 publications

Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C)

Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C)

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases

Enantiocomplementary Michael Additions of Acetaldehyde to Aliphatic Nitroalkenes Catalyzed by Proline‐Based Carboligases

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