Aplicación de Indicador para evaluar la Eficacia

The next step towards the creation of an enzyme equipped with a catalytic amino acid is to identify potential biomolecular scaffolds. The scaffold chosen should meet several criteria: (1) An ideal scaffold will be highly stable. Evolution can often lead to reduced protein stability due to deleterious mutations. We therefore focused on thermophilic proteins which operate at temperatures between 50 and 112 °C. Starting with thermophilic proteins increases the mutation potential without

compromising stability. In addition, we focused our scaffold choices to proteins that are known to (2) express well in E. coli and (3) that contain large enough pockets to spatially accommodate the unnatural amino acid. Finally, we refined our search criteria to include (4) proteins that have previously been used as scaffolds for engineering since they are often well characterized and robust to mutagenesis.

Scaffolds were chosen based on previous successes in the engineering of metalloenzymes and computationally designed enzymes. Scaffolds with large

hydrophobic cores such as the α/β barrels and jellyrolls are popular choices (36, 46). Three enzymes that adopt an α/β barrel fold, 2-deoxyribose-5-phophate aldolase (DERA), indole-3-glycerol phosphate synthase (IGPS) and a synthase subunit of the

glutaminase synthase enzyme complex (tHisF) were selected. Mechanistically, DERA represents a privileged scaffold in aldolase chemistry. It is the only aldolase to catalyze the reaction between two aldehyde substrates. IGPS is an interesting scaffold that contains a wide opening on one side of the α/β barrel, termed the catalytic face. This face has been used in the design of a copper-binding site which was used in a catalytic Diels-Alder cycloaddition in the related imidazole-3-glycerol phosphate synthase. The authors were able to make seven mutations to the scaffold including four mutations of histidine to alanine without any negative effects in

expression and folding (89). In the Baker lab, IGPS has been computationally remodeled as a retro-aldolase and DERA was redesign as a Kemp eliminase (53, 90). Both non-natural activities were improved upon using directed evolution (91). tHisF has been demonstrated as a robust host for the creation of artificial

metalloenzymes and a suitable host for bulky organocatalysts. It is also very tolerant to mutations and simply purified from endogenous proteins by heat denaturation (35, 92). The genes encoding these proteins were obtained from LifeTechnologies as GeneArt Strings DNA Fragments and cloned into the pET21C(+) using the NdeI and XhoI restriction enzyme sites. The genes were amplified with primers containing NcoI and PmeI and subsequently digested and ligated into pBad_pylT backbone, see Primer Sequences 2.5.2.

Scaled preparation of the scaffold proteins was carried out followed by evaluation of aldol catalysis in the model reaction between acetone and 4-

nitrobenzaldehyde. The protein host for the catalytic unnatural amino acid should not perform the reaction on its own. Both IGPS and DERA displayed significant product

formation in the reaction as judged by HPLC analysis of the reaction in the presence of 5 mol% protein, 10 mM aldehyde and 100 mM acetone in phosphate buffer, Figure 2.12. Therefore, ProK engineering efforts with these two scaffolds will require prior mutagenesis to knock out this background catalysis. Wild type tHisF did not catalyze the reaction above background in the absence of catalyst and could be moved forward to ProK incorporation directly.

Figure 2.12 Evaluation of aldol activity in wild type scaffold proteins

HPLC traces of the reaction between 4-nitrobenzaldehyde and acetone with 5 mol% scaffold protein in optimized buffered conditions (A) DERA, PDB ID: 2UB3, demonstrates near complete conversion of the model aldol reaction. (B) tHisF, PDB ID: 1THF, does not catalyze the model aldol reaction. (C)

IGPS, PDB ID: 1CV4, is efficient at the model aldol reaction.

The aldolase scaffold, DERA, is known to catalyze aldol reactions between various ketones and aldehydes, yet this is first example of catalysis with a

500 1500 2500 3500 5.0 8.3 11.7 5 mol %DERA 500 1500 2500 3500 5.0 8.3 11.7 1000 2000 3000 4000 5.8 7.5 9.2 10.8 negative 5 mol % tHisF negative 5 mol % IGPS O OH NO2 NO2 H O

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substituted aromatic acceptor substrate (93). DERA has two active site lysine

residues (K151 and K180) that are crucial for its wild type chemistry, the conversion of 2-deoxy-D-ribose 5-phosphate to D-glyceraldehyde 3-phosphate and

acetaldehyde. This reaction proceeds via a organocatalysis mechanism that requires the nucleophilic attack of lysine 151 on the aldehyde of the substrate. The

nucleophilicity of K151 is made possible by the pKa perturbation in response to the

second lysine residue, K180, which sits adjacent to K151. We hypothesized that these two lysines are the primary source for the background catalysis in our model reaction. To test this hypothesis, either the catalytic lysine or the pKa perturbing

lysine was individually mutated to alanine and both alanine mutations eliminated aldol chemistry between acetone and 4-nitrobenzaldehyde, Figure 2.13.

A similar rational targeted mutagenesis effort to eliminate promiscuous aldol catalysis by IGPS was not undertaken. The native IGPS reaction, conversion of 1-(o- carboxyphenylamino)-1-deoxyribulose 5-phosphate into indole-3-glycerol phosphate, is catalyzed by an intricate interplay of several residues and dependent on

conformation loop changes (94). Based on the complexity of this system, multiple mutations may be required to completely attenuate background catalysis and identifying rational mutations is not straight forward. Therefore, this protein was removed from future work. Since tHisF shows no aldol catalysis and we have

identified point mutants of DERA that are also clear of background chemistry; these two protein scaffolds were selected for subsequent ProK-catalysis experiments.

Figure 2.13 Elimination of WT-DERA aldol catalysis