Altering the substrate specificity of 2-keto-3-deoxy-6-phosphogluconate aldolase by rational mutagenesis and directed evolution.

The need to synthesize enantiomerically pure pharmaceutical, agrochemical, and food agents, has prompted our efforts to develop stereospecific aldol catalysts using 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. This enzyme catalyzes a highly stereoselective carbon-carbon bond forming aldol addition reaction between pyruvate and D-glyceraldehyde-3-phosphate. To engineer aldol catalysts with unnatural substrate specificity, we under took (1) mutational analysis of active-site residues to understand the interactions which are important for molecular recognition, (2) structure base-redesign to alter the substrate specificity, and (3) directed evolution to identify mutants with enhance catalytic activity for hydrophobic aldehydes. In this thesis we report the effects of alanine mutations at R49 and T73 of Escherichia coli KDPG aldolase. These mutations cause significant decreases in the catalytic activity consistent with these residues playing important roles in substrate binding and proper orientation for catalysis. These findings suggest that orientation of the substrate is critical for efficiently catalytic function of E. coli KDPG aldolase, and are consistent with our mechanistic proposal that the deprotonation steps in KDPG aldolases are mediated by an active site water molecule. We also report an in vivo selection methodology which always for the rapid screening of up to 107 members for increased catalytic efficiency for aldol cleavage of novel aldehydes. We used this selection methodology in combination with structure based-redesign to engineer an aldolase which enhances the rate of catalysis of 2-keto-4-hydroxy-(2'-pyridyl)-butyrate (S-KHPB) by 440-fold. This mutant retains high stereoselectivity and has a kcatS -KHPB that is 3-times faster than the rate of cleavage of the natural substrate using the wild-type enzyme. This mutant has broad specificity, retaining 20% of wild-type activity with the KDPG sugar and increases the cleavage of KHO by 51-fold. This mutant is a valuable addition to the available enzymatic aldol catalysts because of its novel specificity for non-carbohydrate molecules. The data in this thesis present useful catalysts for important synthetic reactions, and lays the ground work for further elaboration of the KDPG aldolase scaffold for use as a catalyst for aldol chemistry.Ph.D.BiochemistryBiological SciencesMolecular biologyOrganic chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126646/2/3276114.pd

The <i>Thermococcus kodakaraensis</i> Tko CDC21-1 Intein Activates Its N-Terminal Splice Junction in the Absence of a Conserved Histidine by a Compensatory Mechanism

Inteins and other self-catalytic enzymes, such as glycosylasparaginases and hedgehog precursors, initiate autocleavage by converting a peptide bond to a (thio)­ester bond when Ser, Thr, or Cys undergoes an N–[S/O] acyl migration assisted by residues within the precursor. Previous studies have shown that a His at position 10 in intein Block B is essential for this initial acyl migration and N-terminal splice junction cleavage. This His is present in all inteins identified to date except the <i>Thermococcus kodakaraensis</i> Tko CDC21-1 intein orthologs and the inactive <i>Arthrobacter</i> species FB24 Arth_1007 intein. This study demonstrates that the Tko CDC21-1 intein is fully active and has replaced the lost catalytic function normally provided by the Block B His using a compensatory mechanism involving a conserved ortholog-specific basic residue (Lys₅₈) present outside the standard intein conserved motifs. We propose that Lys₅₈ catalyzes the initial N–S acyl migration by stabilizing the thiazolidine–tetrahedral intermediate, allowing it to be resolved by water-mediated hydrolysis rather than by protonating the leaving group as His is theorized to do in many other inteins. Autoprocessing enzymes may have more flexibility in evolving catalytic variations because high reaction rates are not required when performing single-turnover reactions on “substrates” that are covalently attached to the enzyme. Consequently, inteins have more flexibility to sample catalytic mechanisms, providing insight into various strategies that enzymes use to accomplish catalysis

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals