Natural history of the E1-like superfamily: implication for adenylation, sulfur transfer, and ubiquitin conjugation

Abstract

The E1-like superfamily is central to ubiquitin (Ub) conjugation, biosynthesis of cysteine, thiamine, and MoCo, and several secondary metabolites. Yet, its functional diversity and evolutionary history is not well understood. We develop a natural classification of this superfamily and use it to decipher the major adaptive trends occurring in the evolution of the E1-like superfamily. Within the Rossmann fold, E1-like proteins are closest to NAD(P)/FAD-dependent dehydrogenases and S-AdoMet-dependent methyltransferases. Hence, their phosphotransfer activity is an independent catalytic "invention" with respect to such activities seen in other Rossmannoid folds. Sequence and structure analysis reveals a striking diversity of residues and structures involved in adenylation, sulfotransfer, and substrate binding between different E1-like families, allowing us to predict previously uncharacterized functional adaptations. E1-like proteins are fused to several previously undetected domains, such as a predicted sulfur transfer domain containing a novel superfamily of the TATA-binding protein fold, different types of catalytic domains, a novel winged helix-turn-helix domain and potential adaptor domains related to Ub conjugation. On the basis of these fusions, we develop a generalized model for the linking of E1 catalyzed adenylation/thiolation with further downstream reactions. This is likely to involve a dynamic interplay between the E1 active sites and diverse fused C-terminal domains. We also predict participation of E1-like domains in previously uncharacterized bacterial secondary metabolism pathways, new cysteine biosynthesis systems, such as those associated with archaeal O-phosphoseryl tRNA, metal-sulfur cluster assembly (e.g., in nitrogen fixation) and Ub-conjugation. Evolutionary reconstructions suggest that the last universal common ancestor contained a single E1-like domain possessing both phosphotransfer and thiolating activities and participating in multiple sulfotransfer reactions. The E1-like superfamily subsequently expanded to include 26 families clustering into three major radiations. These are broadly involved in Ub activation, cofactor and cysteine biosynthesis, and biosynthesis of secondary metabolites. In light of this, we present evidence that in eukaryotes other E1-like enzymes such as Urm1 were independently recruited for Ubl conjugation, probably functioning without conventional E2-like enzymes.

Figure 1. Evolutionary history and contextual information for the E1 superfamily

Individual families are listed to the right of the diagram with solid horizontal lines tracing the inferred evolutionary depth of each family across several key evolutionary transition events represented by the labeled vertical lines. Horizontal lines are color-coded (see key) by observed phyletic distributions. Horizontal lines connecting to a dashed ellipse indicates the family descended from any one of the lineages bundled by the ellipse. Colored circles placed at points along the horizontal lines indicate loss of an ancestral sequence feature in lineage (see key at the bottom of the figure). Representative domain architectures and conserved gene neighborhoods of the families are shown to the right of the family name. Colored polygons represent individual protein domains, while boxed arrows represent individual genes in conserved gene neighborhoods. Breaks within a domain indicate a loss of one or more structural elements. Inactive domains are marked with an ‘X’. General functional roles of the different families are listed to the right. Abbreviations: Rhod., Rhodanese domain; CCTBP, Cysteine-containing TBP-like domain; AOR, Aldehyde ferredoxin oxidoreductase; desulf., desulfurase; FMN red., flavindependent oxidoreductase; Pept., peptidase; GNAT, GNAT-type acetyltransferase; ABC_t, ABC transporter; X, predicted novel peptidase domain; Y, predicted metal-binding domain.

Figure 2. Topology diagrams and comparison with other Rossmann-like proteins

Representative cores of Rossmann-like domains belonging to different classes of the fold are depicted as cartoons. Inserts and other lineage-specific features are depicted and labeled with various other colors. Gray spheres represent the magnesium ions in various active sites. Residues experimentally shown to contribute to catalytic activity in the given representatives are labeled. Strand numbers are given at the bottom of each strand; “eq” refers to strands that are spatially equivalent to strands in the E1 superfamily.

Figure 3. Phylogenetic tree of the ThiF/MoaD/MOCS3 family along with along with domain architectures and gene neighborhoods

The tree was constructed using the least-squares method followed by local rearrangement to obtain a maximum-likelihood tree. Closely-related branches have been combined into groups, whose sizes are scaled relative to the number of lineages within each group. Nodes in the tree with >70% bootstrap support are denoted by small gray circles. Groups are colored according to associations with the rhodanese and CCTBP domains (see key at bottom left). The taxonomy of the lineages within a group is also indicated (see key at bottom right). Neighborhoods and architectures are as in Fig. 1; genes encoding multi-domain proteins are shown as boxed arrows demarcated by horizontal lines. Additional abbreviations not found in Figure 1: Ubl, ubiquitin-like; CT_p, C-terminal processing serine peptidase; CS, cysteine synthase, MS, methionine synthase.

Figure 4. Multiple sequence alignment of CCTBP-like and TATA-box binding protein domains

Proteins are labeled to the left of each sequence by their gene names, species abbreviations, and gi numbers demarcated by underscores. Amino acid residues are colored according to side chain properties and degree of conservation within the alignment, set at 75% consensus. The secondary structure is indicated above the alignment. The conserved cysteine of the CCTBP domain is in yellow and shaded in red. The consensus abbreviations and coloring scheme are as follows: h, hydrophobic residues shaded yellow; s, small residues colored green; p, polar residues colored purple; +, positively charged residues colored blue and shaded gray; and b, big residues colored blue. The conserved glycine residue is colored light green. Species abbreviations are as follows: Abac: Acidobacteria bacterium; Aful: Archaeoglobus fulgidus; Amet: Alkaliphilus metalliredigenes; Anid: Aspergillus nidulans; Apen: Acetabularia peniculus; Aper: Aeropyrum pernix; AtCV1: Acanthocystis turfacea Chlorella virus 1; Atha : Arabidopsis thaliana; Bfir: Bacillus firmus; Blic: Bacillus licheniformis; Bmar: Blastopirellula marina; Bsp.: Bacillus sp.; Bsub: Bacillus subtilis; Bthu: Bacillus thuringiensis; CDes: Candidatus Desulfococcus; CKue: Candidatus Kuenenia; Cele: Caenorhabditis elegans; Cmaq: Caldivirga maquilingensis; Csym: Cenarchaeum symbiosum; Esib: Exiguobacterium sibiricum; Gkau: Geobacillus kaustophilus; Glam: Giardia lamblia; Hasp: Halobacterium sp.; Haur: Herpetosiphon aurantiacus; Hmob: Heliobacillus mobilis; Hsap: Homo sapiens; Linn: Listeria innocua; Lmon: Listeria monocytogenes; Lpla: Lactobacillus plantarum; Lreu: Lactobacillus reuteri; Mace: Methanosarcina acetivorans; Maeo: Methanococcus aeolicus; Mbar: Methanosarcina barkeri; Mbur: Methanococcoides burtonii; Mjan: Methanocaldococcus jannaschii; Mlab: Methanocorpusculum labreanum; Mmar: Methanoculleus marisnigri; Mmaz: Methanosarcina mazei; Mmus: Mus musculus; Moth: Moorella thermoacetica; Mthe: Methanosaeta thermophila; Mthe: Methanothermobacter thermautotrophicus; Mthe: Methanothermococcus thermolithotrophicus; Nmar: Nitrosopumilus maritimus; Npha: Natronomonas pharaonis; Nvec: Nematostella vectensis; Oluc: Ostreococcus lucimarinus; Paby: Pyrococcus abyssi; PbCVN : Paramecium bursaria Chlorella virus NY2A; Pfur: Pyrococcus furiosus; Pthe: Pelotomaculum thermopropionicum; Ptor: Picrophilus torridus; Pyae: Pyrobaculum aerophilum; Rbal: Rhodopirellula baltica; Saur: Staphylococcus aureus; Scer: Saccharomyces cerevisiae; Sepi: Staphylococcus epidermidis; Sfum: Syntrophobacter fumaroxidans; Shae: Staphylococcus haemolyticus; Smar: Staphylothermus marinus; Spom: Schizosaccharomyces pombe; Stok: Sulfolobus tokodaii; Susi: Solibacter usitatus; Taci: Thermoplasma acidophilum; Tkod: Thermococcus kodakarensis; Tneu: Thermoproteus neutrophilus; Umet: uncultured methanogen; Usul : uncultured sulfate-reducer.