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.