Redesigning Aldolase Stereoselectivity by Homologous Grafting

Abstract

The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity.

Fig 1

The natural reaction of the enantiocomplementary pyruvate–dependent aldolases KDPG and KDPGal (top) and of DERA (bottom). An enzyme that is diastereoselectively complementary to DERA (“DEXA” = 2-deoxy-d-xylose-5-phosphate aldolase) does not exist in nature as indicated by the dashed line.

Fig 2. Top view on KDPGal (PDB ID: 2V82) [17].

The substrate is highlighted in stick representation. We hypothesize that the blue part of the protein is adapted to fix the nucleophilic carbonyl (here pyruvate), whereas the orange part is adapted to the electrophilic carbonyl (here glyceraldehyde-3-phosphate) and, thus, provides the basis for stereoselectivity.

Fig 3. Geometric analyses of homologous aldolases.

(A) – Superposition of DERA (PDB ID: 1JCL, blue) [37], KDPG (PDB ID: 1EUA, orange) [16], and KDPGal (PDB ID:2V82, green) [17] illustrates the structural similarity of these TIM-barrel proteins. (B-D) – Close-up views of the active sites of the three proteins. The active lysine is marked by a triangle (▼). (E) – Superposition of the active sites of KDPG and KDPGal aldolase. Atoms within a radius of 6 Å around the newly formed stereogenic center are indicated as spheres. (F) – Distances from MD simulations between the ε-amino group of the active lysine and all atoms within the 12 Å-Ø-sphere around the newly formed stereogenic center for both the KDPG and KDPGal aldolase, respectively. The mean values over all conformations of the MD simulation are given; bars indicate the standard deviation.

Fig 4. Phylogenetic analyses of the β-strands 1 and 7 from bacterial KDPG and KDPGal aldolases.

The phylogenetic tree has been created using iTOL and shows the phylogenetic distribution KDPG/KDPGal-sequences used for identifying the respective patterns (~6000 sequences) [42]. The sequence logos have been created using the WebLogo server [43, 44]. The overall height of the stack indicates the sequence conservation at that position, while the height of individual symbols indicates the relative frequency of each amino acid at that position.

Fig 5. Model aldol reaction for testing the stereoselectivity of KDPG, KDPGal, and specific mutants.

The aldol reaction of propanal (1) and pyruvic acid (2) leads to the enantioselective generation of 3 and, by acidic lactonization, to butyrolactone 4.

Fig 6

Principle of KDPG, KDPGal, and DERA-catalyzed reactions for library screening (a) KDPG and KDPGal aldolase, 275 mM propanal, 20 mM KP_i, 1 M sodium pyruvate, pH 6.5, 30°C, 200 rpm, 18 h. (b) Acidified with sulfuric acid to pH 1. (c) 0.1 mmol propanal, 1.3 eq. acetaldehyde, 20 μl DERA crude extract, 0.5 ml triethanol amine buffer (0.1 M, pH7), 5% (v/v) dimethyl sulfoxide, 25°C, 200 rpm, 18 h. (d) 0.5 ml dimethyl sulfoxide, 2 eq. 2,4-dinitrophenylhydrazine, 3 eq. conc. HCl, 50°C, 2 h, 300 rpm.

Fig 7. Enantiomeric excesses of the first generation variants of DERA at sites T18, L20, and A203, respectively.

The ee was determined by the aldol screening (Fig 6) using chiral HPLC. The error bars represent the standard deviation of triplet measurements. A negative control without enzyme did not show any product formation. “*”: Results for variants with an activity lower than 2.0% of the DERA wt were omitted.

Fig 8. Comparison of DERA wt (blue) and the variant A203G ΔG204 ΔG205 (grey).

The sites G204 and G205 are highlighted in red in the wt structure. The models were generated based on PDB ID 1JCL [37] using USCF Chimera [52].

Fig 9. Enantiomeric excesses of the second generation of variants of DERA at sites T18 combined with A203G, A203G ΔG204, and A203G ΔG204 ΔG205, respectively.

The ee was determined by the aldol screening (Fig 6) using chiral HPLC. The error bars represent the standard deviation of triple measurements. A negative control without enzyme did not show any product formation. “*”: Results for variants with an activity lower than 2.0% of the DERA wt were omitted.