Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant.

One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases.

A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System.

Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS).

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography.

Generating random circular permutation libraries.

Protein engineering by random circular permutation is an effective tool for tailoring protein topology with potential functional benefits including improved catalytic activity. This method involves covalently connecting the native protein termini with a peptide linker and cleaving a peptide bond elsewhere in the polypeptide sequence. Termini relocation can impact protein ternary and quaternary structure and translate into functional enhancements due to changes in protein conformation and flexibility.

Truncated FAD synthetase for direct biocatalytic conversion of riboflavin and analogs to their corresponding flavin mononucleotides.

The preparation of flavin mononucleotide (FMN) and FMN analogs from their corresponding riboflavin precursors is traditionally performed in a two-step procedure. After initial enzymatic conversion of riboflavin to flavin adenine dinucleotide (FAD) by a bifunctional FAD synthetase, the adenyl moiety of FAD is hydrolyzed with snake venom phosphodiesterase to yield FMN. To simplify the protocol, we have engineered the FAD synthetase from Corynebacterium ammoniagenes by deleting its N-terminal adenylation domain.

Improved biocatalysts from a synthetic circular permutation library of the flavin-dependent oxidoreductase old yellow enzyme.

Members of the old yellow enzyme (OYE) family are widely used, effective biocatalysts for the stereoselective trans-hydrogenation of activated alkenes. To further expand their substrate scope and improve catalytic performance, we have applied a protein engineering strategy called circular permutation (CP) to enhance the function of OYE1 from Saccharomyces pastorianus. CP can influence a biocatalyst's function by altering protein backbone flexibility and active site accessibility, both critical performance features because the catalytic cycle for OYE1 is thought to involve rate-limiting conformational changes.

Novel protease inhibitors via computational redesign of subtilisin BPN' propeptide.

The propeptide domain of subtilisin BPN' functions as a molecular chaperone for its cognate protease yet quickly assumes a predominantly unfolded structure following cleavage by the mature protease. In contrast, structural stabilization of the propeptide domain has been proposed to competitively inhibit protease self-cleavage, suggesting the possibility for the generation of novel proteinaceous subtilisin inhibitors. Using a Rosetta fixed backbone design, we have redesigned the subtilisin BPN' propeptide structure to generate synthetic peptide sequences with increased and tunable structural stability.