Identification of catalytic amino acids in the human GTP fucose pyrophosphorylase active site.

GTP-l-fucose pyrophosphorylase(GFPP) catalyzes the reversible formation of the nucleotide-sugar GDP-beta-l-fucose from guanosine triphosphate and beta-l-fucose-1-phosphate. The enzyme functions primarily in the mammalian liver and kidney to salvage free fucose during the breakdown of glycoproteins and glycolipids. GFPP shares little primary sequence identity with other nucleotide-sugar metabolizing enzymes, and the three-dimensional structure of the protein is unknown. The enzyme does contain several sequences that could be nucleotide binding sites, but none of them are an exact match to consensus sequences. Using a combination of site-directed mutagenesis and UV photoaffinity cross-linking, we have identified five amino acid residues that are critical for catalysis. Some of these amino acids are found within the poorly conserved nucleotide binding consensus structures, while others represent new motifs. Two active site lysines can be cross-linked to photoaffinity probes. The site of cross-linking depends on the probe used. The identification of these critical residues highlights how distinct GFPP is from other nucleotide-sugar pyrophosphorylases.

Substrate discrimination by the human GTP fucose pyrophosphorylase.

GTP-l-fucose pyrophosphorylase (GFPP, E. C. 2.7.7.30) catalyzes the reversible condensation of guanosine triphosphate and beta-l-fucose-1-phosphate to form the nucleotide-sugar GDP-beta-l-fucose. The enzyme functions primarily in the mammalian liver and kidney to salvage free l-fucose during the breakdown of glycolipids and glycoproteins. The mechanism by which this protein discriminates between substrate and nonsubstrate molecules has been elucidated for the first time in this study. The ability of GFPP to form nucleotide-sugars from a series of base-, ribose-, phosphate-, and hexose-modified precursor molecules has revealed that the enzyme active site senses a series of substrate substituents that drive substrate/nonsubstrate discrimination. These substituents alter the ability of the precursor molecule to interact with the enzyme, as measured by either changes in the Michaelis constant, K(m), the binding affinity, K(a), or through changes in enzymatic turnover, k(cat). In this work, the combined substrate binding and enzyme analysis has revealed that the nature of the purine base is the major determinant in substrate specificity, followed by the nature of the hexose-1-P, and finally by the ribose moiety. Binding is enthalpy-driven and does not involve proton transfer. For the majority of nucleotide-sugar analogues, binding to GFPP is entropically unfavorable; however, surprisingly, a few of the substrate analogues tested bind to GFPP with a favorable entropic term.

"Molecular chameleons". Design and synthesis of a second series of flexible nucleosides.

The second series of flexible shape-modified nucleosides is introduced. The "fleximers" feature the purine ring systems split into their individual imidazole and pyrimidine components. This structural modification serves to introduce flexibility to the nucleoside while still retaining the elements essential for recognition. As a consequence, these structurally innovative nucleosides can more readily adapt to their environment and should find use as bioprobes for investigating enzyme-coenzyme binding sites as well as nucleic acid and protein interactions. Their design and synthesis is described.

"Molecular chameleons". Design and synthesis of C-4-substituted imidazole fleximers.

The synthesis of two flexible nucleosides is presented. The "fleximers" feature the purine ring system split into its imidazole and pyrimidine components. This modification serves to introduce flexibility to the nucleoside while still retaining the elements essential for molecular recognition. As a result, these structurally innovative nucleosides can more readily adapt to capricious binding sites and, as such, should find use for investigating enzyme-coenzyme as well as nucleic acid-protein interactions.

Creation and discovery of ligand-receptor pairs for transcriptional control with small molecules.

The nuclear receptor retinoid X receptor (RXR) is a ligand-activated transcription factor. To create receptors for a new ligand, a structure-based approach was used to generate a library of approximately 380,000 mutant RXR genes. To discover functional variants within the library, we used chemical complementation, a method of protein engineering that uses the power of genetic selection. Wild-type RXR has an EC50 of 500 nM for 9-cis retinoic acid (9cRA) and an EC50 of >10 microM for the synthetic retinoid-like compound LG335 in yeast. The library produced ligand-receptor pairs with LG335 that have a variety of EC50 values (40 nM to >2 microM) and activation levels (10-80% of wild-type RXR with 9cRA) in yeast. The variant I268V;A272V;I310L;F313M has an EC50 for LG335 of 40 nM and an EC50 for 9cRA of >10 microM in yeast. This variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at a 10-fold-lower ligand concentration in yeast. This EC50 is 25-fold lower than the best receptor we have engineered through site-directed mutagenesis, Q275C;I310M;F313I. Furthermore, the variants' EC50 values and activation levels in yeast and mammalian cells correlate. This protein engineering method should be extendable to produce other functional ligand-receptor pairs, which can be selected and characterized from libraries within weeks. Coupling large library construction with chemical complementation could be used to engineer proteins that bind virtually any small molecule for conditional gene expression, applications in metabolic engineering, and biosensors and to engineer enzymes through genetic selection.

Conformational properties of shape modified nucleosides--fleximers.

A detailed (1)H NMR conformational study complemented with ab initio computations was performed in solution on fleximer nucleosides 1, 3, and 5 in relation to their natural counterparts. The substitution of the purine nucleobase found in the natural nucleosides with a more flexible two-ring heterocyclic system strongly increased the population of anti conformation around the glycosidic bond. This was accompanied by a large shift toward a north-type sugar conformation, which was explained by the interplay of anomeric, gauche, and steric effects. The formal separation of the bicyclic purine base into its imidazole and pyrimidine moieties allows for formation of a hydrogen bond between the NH(2) and 2'-OH groups and facilitates favorable conjugation between the two heterocyclic rings. Our results show that the interplay of stereoelectronic effects, combined with the flexibility of the nucleobase and possible conjugation effects within the nucleobase, plays a crucial role in the search for shape-mimic nucleosides that will interact with flexible binding sites.

Carbocyclic isoadenosine analogues of neplanocin A.

[structure: see text] Isoadenosine (IsoA), a structural isomer of adenosine, was shown to possess interesting biological activity but was inherently unstable. In an effort to overcome this, we have designed a series of carbocyclic IsoA analogues, combining the unique connectivity of IsoA with the structural features of some biologically significant Neplanocin A analogues. Their design, synthesis, and structural elucidation is reported.

Unexpected inhibition of S-adenosyl-L-homocysteine hydrolase by a guanosine nucleoside.

A series of shape-modified flexible nucleosides ('fleximers', 1, 2, and 3) was modeled, synthesized and subsequently assayed against S-adenosyl-L-homocysteine hydrolase (SAHase). No inhibitory activity was observed for the adenosine fleximer, which served as a substrate, but moderate inhibitory activity was exhibited by the guanosine fleximers. This is the first known report of a guanosine nucleoside analogue possessing activity against SAHase.

Design and synthesis of a series of chlorinated 3-deazaadenine analogues.

A series of chlorinated adenine analogues were designed with sights set on the development of potential antitumor agents. During the synthetic efforts, two unexpected compounds were identified. Their synthesis, along with synthesis of the chlorinated targets is presented herein.

"Fleximers". Design and synthesis of a new class of novel shape-modified nucleosides(1).

A new class of shape-modified nucleosides is introduced. These novel "fleximers" feature the purine ring systems of adenosine, inosine, and guanosine split into their individual imidazole and pyrimidine components (as in 1-3). This structural modification serves to introduce flexibility into the nucleoside while still retaining the elements essential for recognition. As a consequence, these novel fleximers should find use as bioprobes for investigating enzyme-coenzyme binding sites as well as nucleic acid and protein interactions. Their design and synthesis are described.