Designing Michaelases: exploration of novel protein scaffolds for iminium biocatalysis

Biocatalysis is becoming a powerful and sustainable alternative for asymmetric catalysis. However, enzymes are often restricted to metabolic and less complex reactivities. This can be addressed by protein engineering, such as incorporating new-to-nature functional groups into proteins through the so-called expansion of the genetic code to produce artificial enzymes. Selecting a suitable protein scaffold is a challenging task that plays a key role in designing artificial enzymes. In this work, we explored different protein scaffolds for an abiological model of iminium-ion catalysis, Michael addition of nitromethane into E-cinnamaldehyde. We studied scaffolds looking for open hydrophobic pockets and enzymes with described binding sites for the targeted substrate. The proteins were expressed and variants harboring functional amine groups – lysine, p-aminophenylalanine, or N6-(d-prolyl)-l-lysine – were analyzed for the model reaction. Among the newly identified scaffolds, a thermophilic ene-reductase from Thermoanaerobacter pseudethanolicus was shown to be the most promising biomolecular scaffold for this reaction.

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SDS-PAGE analysis
For SDS-PAGE analysis of all samples a PageRuler™ Prestained Protein Ladder (Thermo Scientific™) marker from 10 to 180 kDa was used.For all SDS-PAGEs, abbreviations stands for: M, Marker; CE, cell-extract; CFE, cell-free extract; FT, flow-through; W, wash with 40 mM imidazole, E, elution fraction and C, concentrated sample, IP, input.Please do not adjust margins Please do not adjust margins For comparison, LmrR (black line), described as dimer ca.30 kDa, [21] was evaluated in the same conditions.Please do not adjust margins Please do not adjust margins  Please do not adjust margins Please do not adjust margins

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Please do not adjust margins Please do not adjust margins Please do not adjust margins  Please do not adjust margins Please do not adjust margins  Please do not adjust margins Please do not adjust margins   Please do not adjust margins Please do not adjust margins Figure S1.Pocket-guided search of scaffolds.LmrR and QacR were used as sequence templates.After an initial search, aberrant sequences were manually discarded.Redundant sequences were discarded with a 90% cut-off.Sequence diversity was analyzed by a cladogram.Templates are indicated with a blue arrow.

Figure S4 .
Figure S4.Size-exclusion chromatography of BbmrR.The oligomeric state of BbmrR (red line) was determined via analytical size-exclusion chromatography on a Superdex 200 10/300 gel filtration column.For comparison, LmrR (black line), described as dimer ca.30 kDa,[21]  was evaluated in the same conditions.

Figure S5 .
Figure S5.Multiple sequence alignment.Preliminary amino acidic sequence analysis was performed using the sequence of LmrR and a selected set of LmrR-like proteins.Analysis was performed MUltiple Sequence Comparison by Log-Expectation (MUSCLE) algorithm using Geneious Prime® 2021.2.2. with ClustalW and UPGMB iteration methods.

Figure S12 .
Figure S12.Preliminary small-scale conversion optimization.a) Reactions were set at pH 5.5-11 in 50 mM HEPES 150 mM NaCl solution.b) Qualitative representation of reaction using 25 μM of pyrrolidine or aniline as side-chain catalysts controls.Reactions were formulated with 1 mM E-cinnamaldehyde and 50 mM nitromethane.

Figure S15 .
Figure S15.TOYE as biocatalyst for Michael addition reaction.Chromatograms of small-scale conversions were performed using 25 μM TOYE, a) and b) I67DProK or c) and d) Y168 DProK at pH 6.5 in 50 mM HEPES and 150 mM NaCl buffer.Reactions were set with 1 mM Ecinnamaldehyde and a) and c) 50 or b) and d) 100 mM nitromethane as nucleophile.Chromatograms were manually shifted to facilitate qualitative comparison.

Figure S17 .
Figure S17.Analytics of substrate and product.Synthesis of 4-nitro-3-phenylbutanal was performed using 5 μM DERA-MA enzyme as a biocatalyst in a 100 mL small-scale reaction.The reaction was formulated with 5 mM E-cinnamaldehyde, 50 mM nitromethane, in 50 mM HEPES, 150 mM NaCl, and 10% ethanol, pH 6.5.The product was obtained after flash chromatography with a 50% yield.The sample was analysed by a) GC and b) NMR.Calibration curves were used to calculate substrate depletion and E-cinnamaldehyde in the product sample.c) A calibration curve using commercial (Merck) E-cinnamaldehyde was used to calculate the remnant substrate obtained in the biosynthesis, then d) the calibration curve of the product was recalculated accordingly.Chromatograms for each concentration of substrate and product, and the residual plots of the calibration curves are shown.

Amino Acid Sequences of Selected Scaffolds
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margins Supporting tableTable S1 .
List of primers used in this study.Mutations are highlighted in red.
TTG CAG CAC CAT TGT TCC This journal is © The Royal Society of Chemistry 2024 Please do not adjust margins Please do not adjust margins