Reconstruction of ancestral metabolic enzymes reveals molecular mechanisms underlying evolutionary innovation through gene duplication

Abstract

Gene duplications are believed to facilitate evolutionary innovation. However, the mechanisms shaping the fate of duplicated genes remain heavily debated because the molecular processes and evolutionary forces involved are difficult to reconstruct. Here, we study a large family of fungal glucosidase genes that underwent several duplication events. We reconstruct all key ancestral enzymes and show that the very first preduplication enzyme was primarily active on maltose-like substrates, with trace activity for isomaltose-like sugars. Structural analysis and activity measurements on resurrected and present-day enzymes suggest that both activities cannot be fully optimized in a single enzyme. However, gene duplications repeatedly spawned daughter genes in which mutations optimized either isomaltase or maltase activity. Interestingly, similar shifts in enzyme activity were reached multiple times via different evolutionary routes. Together, our results provide a detailed picture of the molecular mechanisms that drove divergence of these duplicated enzymes and show that whereas the classic models of dosage, sub-, and neofunctionalization are helpful to conceptualize the implications of gene duplication, the three mechanisms co-occur and intertwine.

Figure 1. Yeast species can grow on a broad spectrum of α-glucosides.

Serial dilutions of each species were spotted on medium (Yeast Nitrogen Base w/o amino acids) with 2% of each sugar (Me-a-Glu  =  methyl-α-glucoside). Growth was scored after 3 d incubation at 22°C. +, growth; −, no growth. # MALS genes, the number of maltase genes found in each of these strains. Genotypes are listed in Table S5.

Figure 2. Duplication events and changes in specificity and activity in evolution of S. cerevisiae MalS enzymes.

The hydrolytic activity of all seven present-day alleles of Mal and Ima enzymes as well as key ancestral (anc) versions of these enzymes was measured for different α-glucosides. The width of the colored bands corresponds to k_cat/K_m of the enzyme for a specific substrate. Specific values can be found in Table S2. Note that in the case of present-day Ima5, we were not able to obtain active purified protein. Here, the width of the colored (open) bands represents relative enzyme activity in crude extracts derived from a yeast strain overexpressing IMA5 compared to an ima5 deletion mutant. While these values are a proxy for the relative activity of Ima5 towards each substrate, they can therefore not be directly compared to the other parts of the figure. For ancMalS and ancMal-Ima, activity is shown for the variant with the highest confidence (279G for ancMalS and 279A for ancMal-Ima). Activity for all variants can be found in Table S2.

Figure 3. Activities of present-day Mal enzymes in distant fungi correspond well with activities of reconstructed ancestral enzymes.

Basic phylogeny of the MALS gene family with different clades, showing the ancestral bifurcation points (indicated by *). Length of the colored bands corresponds to the measured k_cat/K_m of the enzyme for a specific substrate. Bands for Ima5 represent relative enzyme activity in crude extracts derived from a yeast strain overexpressing IMA5 compared to an ima5 deletion mutant. For ancMalS and ancMal-Ima, activity is shown for the variant with the highest confidence (279G for ancMalS and 279A for ancMal-Ima). Error bars represent standard deviations. Activity for all variants and the corresponding standard deviations can be found in Table S2.

Figure 4. Positive selection on residues near binding pocket resulted in distinct subgroups with different substrate preference.

An unrooted codon-based phylogenetic tree of the MALS gene family is shown on the left. Branches are colored according to the ω (dN/dS) rate classes inferred from GA Branch analysis . Branches for which branch-site tests for positive selection were performed are indicated by colored arrowheads. Since ω rate classes cannot be inferred reliably for very small branches, branches <0.01 are not colored. The right part of the figure shows the nine variable AA residues located near the substrate binding pocket of the respective enzymes (numbering based on Ima1 sequence). Sequences of ancestral enzymes are shaded in grey. Subgroups of enzymes that show similar substrate specificity are colored accordingly. Residues indicated in bold were found to be under positive selection by the branch-site tests. Perfectly co-varying residues are boxed. Substrate preference of extant and ancestral enzymes was deduced from enzyme assays on S. cerevisiae, K. lactis, K. thermotolerans, L. elongisporus, and reconstructed ancestral enzymes (see Figure 3 and Table S4).

Figure 5. Three co-evolving residues determine the shift in activity observed in the evolution of Ima1–4.

(A) Global structure of the MalS proteins with maltose, represented as spheres, bound in the active site. Panels (B–E) show details of the active site, with substrates as sticks (maltose in panels B and C; isomaltose in panels D and E). The variable AAs are shown as spheres. Structural analysis of the binding site suggests that the A279Q mutation affects substrate specificity the most. The side chain of Q279 sterically hinders binding of maltose but stabilizes isomaltose binding through polar interactions. The G218S and V278M changes cause subtle adaptations of the fold, causing Q279 to protrude further into the binding pocket, which allows optimal interaction with isomaltose. (F) Activity (k_cat/K_m) of all possible intermediary forms in the evolution of three co-evolving residues in AncIma1–4, obtained from enzyme assays performed for all reconstructed proteins. Values for k_cat and K_m can be found in Table S2.

Figure 6. Evolution of the promiscuous AncMalS enzyme into isomaltose- and maltose-hydrolyzing enzymes.

AncMalS is a promiscuous enzyme that hydrolyzes both maltose- and isomaltose-like substrates, whereas the present-day enzymes Ima1,2 and Ima5 preferentially hydrolyze isomaltose-like sugars and Mal12–32 preferentially hydrolyzes maltose-like sugars. First, the presence of a Thr or Val residue at position 216 affects the binding affinity of the enzyme through changes in the hydrophobic/hydrophilic interactions with the different substrate classes (panels A to D; see also Figure S8). The case of Ima1,2 and Ima5 (panels C to F) illustrates that an additional shift in substrate specificity can be obtained via different evolutionary routes. In the case of Ima1 and Ima2, the change of G279 to Q279 interferes with binding of maltose-like substrates, but the side chain of Gln can undergo polar interactions with isomaltose (panels C and D). The G218S and V278M changes cause additional subtle adaptations of the protein fold, causing Q279 to protrude further into the binding pocket, allowing optimal interaction with isomaltose (see also Figure 5). The evolution of isomaltase activity in Ima5 also occurred via the introduction of steric hindrance in the binding pocket, although in this case the change involved was L219M (panels E and F). In ancMalS, residues D307 and E411 allow binding of both maltose- and isomaltose-like substrates (panels G and H). In the maltose-specific enzymes Mal12 and Mal32, however, these residues have evolved to E307 and D411 (panels I and J). These changes not only increase the affinity for maltose-like substrates but also make this site incompatible with isomaltose-like substrates. Subpanels are graphical representations of the binding pocket, with key amino acids depicted as spheres. Maltose and isomaltose are represented as sticks.

Figure 7. Multiple evolutionary mechanisms contribute to the evolution of the MalS gene family in S. cerevisiae.

(A) Overview of evolutionary mechanisms in the evolution of an ancestral gene with two conflicting activities (major function, red; minor function, blue). Duplication can help resolve this “adaptive conflict” by allowing optimization of these activities in two separate copies. Increased requirement for either of these activities, for example by changes in the environment, can first be met by duplication of the ancestral gene. Selection for increased gene dosage can help to preserve both copies until adaptive mutations optimize the different functions in separate copies. (B) Evolution of the promiscuous ancestral MalS enzyme into the seven present-day MalS alleles shows how different evolutionary forces contribute to the evolution of gene duplicates. Activity towards isomaltose-like sugars first existed only as a trace activity in the ancestral, preduplication enzyme. The nature of the binding pocket prevented simultaneous optimization of the major and minor function in the ancestral enzyme. Duplication allowed the (full) optimization of the two conflicting activities of the ancestral enzyme in separate copies. Several key residues in the enzymes' binding pocket responsible for these shifts in substrate specificity (shaded in grey) show signs of positive selection (indicated both in red and with red arrows; see also Figure 4). Preservation of more recent, highly similar duplicates like Mal12–Mal32 may be mediated through gene dosage effects (see also Figure S9). Sequences above each enzyme represent the nine variable residues in the binding pocket (numbering based on Ima1 sequence). AA changes that led to improvement of one of the hydrolyzing activities are shaded in grey.