Expanding the synthetic utility of KDPG and KDPGal aldolases.

摘要

We report the investigations of a three-pronged approach to expanding the synthetic utility of two complementary pyruvate aldolases, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase (EC 4.1.2.21). The three approaches address explorations of the synthetic utility of KDPGal aldolase, the utility of KDPG aldolase in the preparation of the amino acid moiety of an antifungal nikkomycin, and the directed evolution of KDPG aldolase with respect to the substrate benzaldehyde.;The first section presents issues relevant to investigations of the synthetic utility of KDPGal aldolase. KDPGal, a required substrate for detection of the corresponding aldolase, was synthesized from D-galactono-1,4-lactone via an improved route in 17% overall yield. KDPGal aldolase was isolated from two original eda- sources, Pseudomonas cepacia and Escherichia coli. The P. cepacia enzyme was purified tenfold in a single differential dye-ligand chromatography step in 84% yield. Stability, pH-activity and substrate specificity studies of the P. cepacia aldolase revealed an enzyme stable to purification exhibiting a relatively wide pH-activity relationship and broad acceptance of a variety of aldehydic substrates. In general, P. cepacia KDPGal aldolase accepts aldehydic substrates with an electron-withdrawing group at either carbons 2, 3, or 4 and shows high specificity for electrophiles with the D configuration at carbon 2. P. cepacia KDPGal aldolase was utilized to synthesize on a preparative scale both 2-keto-3-deoxy-galactonate and (R)4-hydroxy-2-keto-4-(2'-pyridyl)butyrate. The second portion discusses expanding the utility of KDPG aldolase to include the synthesis of a noncarbohydrate product. Specifically, KDPG aldolase was isolated from E. coli via a literature method and utilized in a novel synthesis of a nikkomycin amino acid moiety. Aldolase-catalyzed condensation followed by phenylalanine dehydrogenase-catalyzed reductive amination produced the desired amino acid in 76% overall yield.;The final segment considers efforts toward expanding the synthetic utility of KDPG aldolase via directed evolution. The contiguous active site region of KDPG aldolase was subjected to a novel error-prone PCR protocol, creating a library of 1 x 104 mutant enzymes. The library was assayed for increased activity with the acceptor benzaldehyde using an alcohol dehydrogenase-based system. Wild-type enzyme accepts benzaldehyde at 10-4 the rate of D-glyceraldehyde-3-phosphate. Three mutants exhibiting 1.3, 1.7, and 2.0 times the activity of the wild-type enzyme were detected in a single generation of random mutagenesis.