No abstract

Ser was replaced at position 286 of firefly luciferase (Luciola mingrelica) by a series of naturally occurring and unnatural amino acids. The effect of these substitutions on the properties of luciferase, such as thermostability, pH dependence, and color of light emitted, was investigated. For these purposes, the Ser286 codon (AGT) was replaced by an amber stop codon (TAG) within the luciferase gene and transformed into Escherichia coli strains producing specific amber suppressor tRNA's to express luciferase with different substitutions at this position. The incorporation of Leu, Lys, Tyr, or Gln at this position reduced the thermostability of mutated luciferases. The color of emitted light changed upon substitution from yellow-green (λmax 582 nm) for the wild-type enzyme having Ser286 to, for example, red (λmax 622 nm) for luciferase having Leu286. For further evaluation of the structural relationship between the amino acid position at 286 and the wavelength of emitted light, we used the method of in vitro incorporation of unnatural amino acids, which involves readthrough of a nonsense (UAG) codon by a misacylated suppressor tRNA. The amino acids incorporated at position 286 in this fashion included O-glucosylated serine, serine phosphonate, tyrosine phosphate, and tyrosine methylenephosphonate. The wavelength of light emitted by the luciferase analogues was measured. While the introduction of serine phosphonate and glucosylated serine did not change the λmax of light produced by luciferase, the incorporation of tyrosine phosphate and tyrosine methylenephosphonate into position 286 altered the spectra of emitted light compared with those of Ser286 and Tyr286. The pH dependence of the wavelength of light emitted by the luciferases containing the negatively charged phosphorylated Tyr analogues was demonstrated and could be rationalized in terms of the pK a's of the phosph(on)ate oxygens.

DNA ligation by DNA topoisomerase I was investigated employing synthetic DNA substrates containing a single strand nick. Site-specific cleavage of the DNA by topoisomerase I in proximity to the nick resulted in uncoupling of the cleavage and ligation reactions of the enzyme, thereby trapping the covalent enzyme−DNA intermediate. DNA cleavage could be reversed by the addition of acceptor oligonucleotides containing a free 5‘-OH group and capable of hybridizing to the noncleaved strand of the “suicide substrates”. Utilizing acceptors with partial complementarity, modification of nucleic acid structure has been obtained. Modifications included the formation of DNA insertions, deletions, and mismatches. To further evaluate the potential of topoisomerase I to mediate structural transformations of DNA, acceptor oligonucleotides containing nucleophiles other than OH groups at the 5‘-end were studied as substrates for the topoisomerase I-mediated ligation reaction. Toward this end, oligonucleotides containing 5‘-thio, amino, and hydroxymethylene moieties were synthesized. Initial investigations utilizing a coupled cleavage−ligation assay suggested that only the modified acceptor containing an additional methylene group underwent efficient enzyme-mediated ligation. However, as linear DNA is not a preferred substrate for topoisomerase I, the enzyme−DNA intermediate was purified to homogeneity, thereby allowing investigation of the ligation reaction independent of the forward reaction that formed the covalent binary complex. The isolated complex consisted of equimolar enzyme and DNA, with topoisomerase I covalently bound to a specific site on the DNA duplex in an enzymatically competent form. Displacement of the enzyme-linked tyrosine moiety of the enzyme−DNA binary complex was effected by all the modified acceptor oligonucleotides, affording unnatural internucleosidic linkages at a specific site. Characterization of the formed linkages was effected both by enzymatic and chemical degradation studies. Comparative analysis revealed overall differences in the efficiency and rate of the topoisomerase I-mediated ligation of the modified acceptors. Moreover, the facility of ligation of the amino acceptor was significantly enhanced at increasing pH values. In addition, the method utilized to obtain the topoisomerase I−DNA intermediate is capable of affording large quantities required for further mechanistic and physicochemical characterization of the formed binary complex.

A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.The "standard model" of nucleic acid structure dates back to 1953 and two classic papers by Watson and Crick.132 It has been little altered since. The model holds that the energy of binding of two complementdry DNA or RNA (oligonucleotide) strands arises from the stacking of the hydrophobic nucleobases, while the specificity of the association arises from base pairing following two simple rules ("A pairs with T, G pairs with C"). No other class of natural products has reactivity that obeys such simple rules. Nor is it obvious how one designs a class of chemical substances that does so much so simply. Despite this chemical conundrum, and the position of nucleic acids at the center of natural product chemistry, few organic chemists have chosen to apply their synthetic skills to explore reactivity at the level of the oligonucleotide. Much work had been done, of course, in making structurally modified analogs of nucleosides, both in industry and academia.3 But most organic chemists, attracted by the structural intricacies of secondary metabolites, have neglected oligonucleotides as targets for structural modification.Some 15 years ago we began a program to fill this gap, developing synthetic organic chemistry and organic structural theory as it applies to nucleic acids in their oligomeric form. This began with one of the first two total syntheses of a gene encoding a p r~t e i n ,~ and has continued with the development of structurally altered oligonucleotides. As in all organic chemistry that alters the structure of natural products, our goal has been to learn more about how DNA and RNA work. We focus here on chemistry that has modified the bases, the sugars, and the backbones of oligonucleotides.

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.