Intramolecular epistasis and the evolution of a new enzymatic function

Abstract

Atrazine chlorohydrolase (AtzA) and its close relative melamine deaminase (TriA) differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine.

Figure 1. Reaction schemes for melamine deaminase (TriA) and atrazine dechlorinase (AtzA).

The hydrolytic deamination of melamine to ammeline by TriA and the dechlorination of atrazine to 2-hydroxyatrazine by AtzA are shown. TriA also possesses a low level of atrazine dechlorinase activity .

Figure 2. Modeled active site of AtzA.

A homology model of the active site of AtzA was used to illustrate the positions of five of the nine amino acid differences between AtzA and TriA. Shown here are the AtzA substrate (atrazine; green), amino acids identical in both AtzA and TriA (Q71, W87, L88, Q96, N126, M155, A216, A220, E246 and D250; white), and amino acids that differ between AtzA and TriA (positions 84, 217, 219, 328 and 331; purple).

Figure 3. Step-wise laboratory-based evolution of AtzA to TriA.

Circles indicate the variants for which the k _cat/K _M values (s⁻¹.M⁻¹; values in Table 1 and Table S2) for atrazine dechlorination and melamine deamination were determined (color coded as follows: AtzA, red (filled); generation 1, orange (filled); generation 2, green (filled); generation 3, blue (filled); generation 4, violet (filled); generation 5, orange (open); generation 6, red (open); generation 7, green (open); generation 8, blue (open); and TriA, violet (open)). Lines are used to link variants differing by one substitution (thick lines link optimal variants; thin lines link the optimal variants to suboptimal variants – suboptimal variants were not used to generate subsequent variants). Amino acid substitutions discussed in the text have been labelled for clarity, as have the wild-type AtzA and TriA enzymes. Inset: expansion of the region of Fig. 3 that contains the last four steps of the trajectory.

Figure 4. Partial step-wise laboratory evolution of TriA to AtzA.

Circles indicate the variants for which the k _cat/K _M values (s⁻¹.M⁻¹; values in Table 1 and Table S2) for atrazine dechlorination and melamine deamination were determined, and are color coded as follows: TriA, red; generation 1, orange; generation 2, green; generation 3, blue; AtzA, violet. Lines are used to link variants differing by one substitution (thick lines link optimal variants; thin lines link the optimal variants to suboptimal variants – suboptimal variants were not used to generate subsequent variants). Amino acid substitutions discussed in the text have been labelled for clarity, as have the wild-type AtzA and TriA enzymes.

Figure 5. Trade-off between atrazine dechlorinase and ametryn hydrolase activity during the transition between AtzA and TriA.

Circles indicate the variants for which the k _cat/K _M values (s⁻¹.M⁻¹; values in Table 1 and Table S2) for atrazine dechlorination and ametryn hydrolysis are shown. Lines are used to link variants differing by a single amino acid. Each variant has been assigned a letter and the identity of the each variant’s direct parent is indicated together with the distinguishing amino acid substitution. The letter assignments correspond to those found in Fig. 3 and Table 1.

Figure 6. Apparent melting temperatures (T_m ^app) of AtzA, TriA and their intermediates.

T_m ^app of the enzyme variants along the step-wise trajectories from AtzA to TriA (A) and from TriA to AtzA (B) calculated from residual enzyme activities after heating for 15 minutes at temperatures between 30°C and 70°C. Each variant has been assigned a letter and the identity of the each variant’s direct parent is indicated together with the distinguishing amino acid substitution. The letter assignments correspond to those found in Figs. 3 and 4 and Table 1. Error bars indicate 95% confidence limits.

Figure 7. Epistatic effects of the C331S substitution in AtzA and the S331C substitution in TriA.

The k _cat/K _M values for atrazine dechlorination (green) and melamine deamination (blue) in the wild-type (dark) or position 331 variant (light) are shown for the substitutions at positions 328 and 84 in AtzA (top) and TriA (bottom). The identity of the amino acid at position 331 (cysteine, C, or serine, S) is indicated for clarity. Error bars indicate standard deviations.

Figure 8. Roles of Amino Acids at Positions 328 and 331 in AtzA and TriA.

In TriA (A) Cys331 donates a proton to the NH₂− leaving group of melamine and abstracts a proton from Asp328. In AtzA (B), the serine hydroxyl group stabilizes the halide of atrazine in the transition state via a hydrogen bonding interaction and is in turn stabilized by Asn328.