Jeffrey V. Miller

Probing Steric and Hydrophobic Effects on Enzyme-Substrate Interactions by Protein Engineering

Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly166), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (kcat/Km) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft (∼160 �3), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (kcat/Km) (up to 5000 times) as a result of steric hindrance.

Production of 2-Keto-L-Gulonate, an Intermediate in L-Ascorbate Synthesis, by a Genetically Modffied Erwinia herbicola

A new metabolic pathway has been created in the microorganism Erwinia herbicola that gives it the ability to produce 2-keto-L-gulonic acid, an important intermediate in the synthesis of L-ascorbic acid. Initially, a Corynebacterium enzyme that could stereoselectively reduce 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid was identified and purified. DNA probes based on amino acid sequence information from 2,5-diketo-D-gluconic acid reductase were then used to isolate the gene for this enzyme from a Corynebacterium genomic library. The 2,5-diketo-D-gluconic acid reductase coding region was fused to the Escherichia coli trp promoter and a synthetic ribosome binding site and was then introduced into E. herbicola on a multicopy plasmid. Erwinia herbicola naturally produces 2,5-diketo-D-gluconic acid via glucose oxidation, and when recombinant cells expressing the plasmid-encoded reductase were grown in the presence of glucose, 2-keto-L-gulonic acid was made and released into the culture medium. The data demonstrate the feasibility of creating novel in vivo routes for the synthesis of important specialty chemicals by combining useful metabolic traits from diverse sources in a single organism.

Elimination of an Immunodominant CD4+ T Cell Epitope in Human IFN-β Does Not Result in an In Vivo Response Directed at the Subdominant Epitope

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4+ T cell epitope peptide region in human IFN-β, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-β molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-β responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-β molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.