title: Potential steps in the evolution of a fused trimeric all-β dUTPase involve a catalytically competent fused dimeric intermediate.
abstract: Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer-like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D. virilis dut gene are not identical due to several point mutations. We investigated the potential evolutionary pathway that led to the emergence of this extant fused trimeric dUTPase in D. virilis. The herein proposed scenario involves two sequential gene duplications followed by sequence divergence amongst the dut repeats. This pathway thus requires the existence of a transient two-repeat-containing fused dimeric dUTPase intermediate. We identified the corresponding ancestral dUTPase single repeat enzyme together with its tandem repeat evolutionary intermediate and characterized their enzymatic function and structural stability. We additionally engineered and characterized artificial single or tandem repeat constructs from the extant enzyme form to investigate the influence of the emergent residue alterations on the formation of a functional assembly. The observed severely impaired stability and catalytic activity of these latter constructs provide a plausible explanation for evolutionary persistence of the extant fused trimeric D. virilis dUTPase form. For the ancestral homotrimeric and the fused dimeric intermediate forms, we observed strong catalytic and structural competence, verifying viability of the proposed evolutionary pathway. We conclude that the progression along the herein described evolutionary trajectory is determined by the retained potential of the enzyme for its conserved three-fold structural symmetry.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. benedek.andras@ttk.mta.hu.|Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Hungary. benedek.andras@ttk.mta.hu.
authors.forename: András
authors.initials: A
authors.lastname: Benedek
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Horváth
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Rita
authors.initials: R
authors.lastname: Hirmondó
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Olivér
authors.initials: O
authors.lastname: Ozohanics
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Angéla
authors.initials: A
authors.lastname: Békési
authors.affiliation: Institute of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
authors.forename: Károly
authors.initials: K
authors.lastname: Módos
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Révész
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Károly
authors.initials: K
authors.lastname: Vékey
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. nagy.gergely@ttk.mta.hu.|Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Hungary. nagy.gergely@ttk.mta.hu.
authors.forename: Gergely N
authors.initials: GN
authors.lastname: Nagy
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. vertessy@mail.bme.hu.|Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Hungary. vertessy@mail.bme.hu.
authors.forename: Beáta G
authors.initials: BG
authors.lastname: Vértessy
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D004330:Drosophila / Q000201:enzymology* / Q000235:genetics*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D019143:Evolution, Molecular; D020440:Gene Duplication; D017344:Genes, Insect; D008958:Models, Molecular; D010802:Phylogeny; D017354:Point Mutation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020080:Tandem Repeat Sequences
chemical_list: D029721:Drosophila Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase
keywords: ancestral reconstitution; dUTPase; enzyme evolution; gene duplication and fusion; gene triplication; oligomeric proteins; protein structure-function relationships