Redesigning Aldolase Stereoselectivity by Homologous Grafting

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

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

<p>The <i>ee</i> was determined by the aldol screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156525#pone.0156525.g006" target="_blank">Fig 6</a>) 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.</p

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

<p>The <i>ee</i> was determined by the aldol screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156525#pone.0156525.g006" target="_blank">Fig 6</a>) 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.</p

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

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

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

<p>The phylogenetic tree has been created using iTOL and shows the phylogenetic distribution KDPG/KDPGal-sequences used for identifying the respective patterns (~6000 sequences) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156525#pone.0156525.ref042" target="_blank">42</a>]. The sequence logos have been created using the WebLogo server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156525#pone.0156525.ref043" target="_blank">43</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156525#pone.0156525.ref044" target="_blank">44</a>]. 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.</p

Michaelis-Menten parameters from steady-state kinetics with 2-deoxy-d-ribose-5-phosphate (D5P) as substrate.

<p>Michaelis-Menten parameters from steady-state kinetics with 2-deoxy-d-ribose-5-phosphate (D5P) as substrate.</p

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

<p>The sites G204 and G205 are highlighted in red in the wt structure. The models were generated based on PDB ID 1JCL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156525#pone.0156525.ref037" target="_blank">37</a>] using USCF Chimera [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156525#pone.0156525.ref052" target="_blank">52</a>].</p

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

<p>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.</p