Crystal structures of two monomeric triosephosphate isomerase variants identified via a directed-evolution protocol selecting for L-arabinose isomerase activity

Abstract

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using an Escherichia coli L-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of the Escherichia coli selection strain in medium supplemented with 40 mM L-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

Keywords: TIM barrel; enzyme engineering; non-natural enzymes; structure-based rational design; triosephosphate isomerase.

Figure 1

Ribbon drawing of the wild-type TIM dimer showing the active-site residues located in loop 1 (Asn11 and Lys13), in loop 4 (His95) and in loop 6 (Glu167). The shortened dimer-interface loops, loop 1, loop 3 and loop 8, are shown in red, orange and green, respectively. Loop 7 is shown in yellow.

Figure 2

The anchoring interactions of Asn11 OD2 in wild-type TIM (cyan; PDB entry 1n55; complexed with PGA in grey) by hydrogen bonds from Asn11 OD2 to Trp12 N and Lys13 N. In the wild-type TIM active site Asn11 ND2 functions as an oxyanion-hole donor for the aldehyde substrate (Kursula et al., 2001 ▸). In A-TIM (green, PDB entry 2vel; complexed with PGA in yellow) the peptide after Trp12 is flipped, causing a different conformation of the Asn11 side chain. Hydrogen-bonding interactions are highlighted by dotted lines.

Figure 3

2F _o − F _c OMIT maps obtained after ten cycles of additional OMIT refinement of the two models, leaving out the mutated side chains and the ligand glycolic acid (GOA). (a) Ma18, near E23G (contour level 1σ). (b) Ma18, near A70T (contour level 0.8σ). (c) Ma18, near S96F (contour level 0.8σ). (d) Ma18, near A178V (contour level 0.8σ). (e) Ma21, near Q65L (contour level 0.8σ). (f) Ma21, near the bound glycolic acid (GOA) (contour level 1σ). The refined models are shown in pink and the A-TIM reference model (PDB entry 2vek) is shown in green.

Figure 4

Ribbon trace of A-TIM (same view as in Fig. 1 ▸; PDB entry 2vel). Highlighted are the five A-TIM residues that were mutated in these experiments: E23G, A70T, S96F and A178V (in Ma18) and Q65L (in Ma21). The active site is identified by the bound active-site ligand PGA, which is shown as a skeleton model.

Figure 5

The loop 1 structure near Asn11 of Ma18 and Ma21. (a) Superposition of Ma18 (blue) and A-TIM (green, complexed with PGA; PDB entry 2vel). (b) Superposition of Ma21 (blue) and A-TIM (pink, complexed with citrate; PDB entry 2vek). In the latter complex the mode of binding of citrate is stabilized by a hydrogen bond to the Asn11 side chain, which is not possible in the Ma21 active site as this side chain is being pushed away by the Leu65 side chain.

Figure 6

Comparison of the modes of binding of glycolate to Ma21 (blue; PDB entry 4pc8), citrate to A-TIM (green, PDB entry 2vek) and PGA to A-TIM (grey; PDB entry 2vel) in the closed loop 6/loop 7 active site. The hydrogen bonds between the carboxylate O atom of glycolic acid (GOA) and Gly173 N (loop 6) and Ser213 N (loop 7) are shown as dotted lines.

Figure 7

The favourable hydrophobic interactions of the Leu65 side chain of Ma21 (PDB entry 4pc8). The dotted lines visualize the carbon–carbon van der Waals contacts of the Leu65 side-chain atoms with protein atoms within 4.4 Å: Asn11 C^β, Lys13 C^β, Ala42 C^β, Thr44 C^γ2, Val92 C^β, Val92 C^γ1 and Val92 C^γ2.