Evolutionary coupling saturation mutagenesis: Coevolution-guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

Abstract

Pullulanases are well-known debranching enzymes hydrolyzing α-1,6-glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis-involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the 'evolutionary coupling saturation mutagenesis' (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.

Keywords: activity; coevolving residues; directed evolution; evolutionary information; pullulanase; saturation mutagenesis.

Figure 1.

Evolutionary coupling saturation mutagenesis (ECSM) strategy (a) using pullulanase from Bacillus naganoensis (b) as a target protein. (a) Schematic representation of ECSM strategy. (b) Domain structure of pullulanase from B. naganoensis: CBM, carbohydrate-binding module; X, domain of unknown function.

Figure 2.

The spatial location in the homology model of BnPUL of the three residue pairs (K631/Q597, V328/I565, D541/D473) identified as mutational hotspots based on EC analysis resulting in mutant enzymes with improved catalytic activity. (a, c) Backbone representation with the X25 and X45 domains shown in teal, the CBM48 domain shown in blue, and the catalytic domain and C-terminal segment shown in gray. The residues in the catalytic triad in the active site of the enzyme (D619, E648, and D733) are shown in purple space-filling representation. The three residue pairs yielding improved catalytic activity are also shown in space-filling representation, with K631/Q597 in green, V328/I565 in yellow, and D541/D473 in red. The view in panel c is rotated by 180˚ from that in panel a. (b, d) Surface representation of BnPUL in the same orientations and with the same color-coding as in panels a and c. These images were all produced using the program PyMOL.

Figure 3.

Flowchart of the two-step saturation mutagenesis performed for two of the seven residue pairs (Table 3). The red plate pullulanase assay is performed after saturation mutagenesis of the first site. Left branch: Compensatory 2nd site mutations identified for 1st site mutations with greatly reduced activity (< ~10% of WT). Right branch: 2nd site mutations for 1st site mutations retaining ~WT activity with significantly increased enzyme activity (Table 3).

Figure 4.

Michaelis-Menten parameters of the seven double mutants (a) and the eight quadruple mutants (b) derived from the evolutionary coupling saturation mutagenesis libraries. The values were measured in triplicate and estimates of the standard deviations are indicated as error bars.

Figure 5.

Interaction between the active residue Asp619 and the targeted α−1,6-glucosidic bond in the pre-reaction state (a) and conformation maps of the highest-efficient mutant and the WT enzyme in the pre-reaction state simulations (b).