Ambar K Choudhury

University of Virginia, Charlottesville, VA, United States

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Publications (3)17.43 Total impact

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    ABSTRACT: Unnatural amino acid mutagenesis provides the wherewithal to study protein function in great detail. To extend the repertoire of functionalized amino acids available for study by this technique, seven structural analogues of arginine were prepared and used to activate a suppressor tRNACUA. These included Ngamma-methylarginine, Ngamma-nitroarginine, citrulline, homoarginine, and three conformationally constrained analogues based on proline. These misacylated tRNAs were shown to be capable of introducing the arginine analogues into dihydrofolate reductase (position 22) and Photinus pyralis luciferase (positions 218 and 437). Most of the modified luciferases containing arginine analogues at position 218 emitted light with less efficiency and at longer wavelength than the wild type. This is consistent with the postulated role of this residue as essential for maintaining the polarity and rigidity of the luciferin-binding site. Interestingly, the luciferase containing Ngamma-methylarginine at position 218 emitted light at the same wavelength as the wild type and was at least as efficient. Alteration of the arginine residue at position 437 had no effect on the wavelength of emitted light but afforded analogues, all of which emitted light less efficiently than the wild type. This is altogether consistent with the putative role of Arg437, which is an invariant residue within the superfamily of enzymes that includes P. pyralis luciferase. This amino acid is part of the linker between the two structural domains of luciferase that is believed to be essential for efficient enzyme function but not part of the substrate-binding site.
    Biochemistry 05/2007; 46(13):4066-76. · 3.38 Impact Factor
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    ABSTRACT: The human topoisomerase I-mediated DNA relaxation reaction was studied following modification of the enzyme at the active site tyrosine (position 723). A series of unnatural tyrosine analogues was incorporated into the active site of human topoisomerase I by utilizing misacylated suppressor tRNAs in an in vitro protein synthesizing system. The relaxation activities of the modified human topoisomerase I analogues having varied steric, electronic, and stereochemical features were all greatly diminished relative to that of the wild type. It was found that modifications involving replacement of the nucleophilic tyrosine OH group with NH2, SH, or I groups eliminated DNA relaxation activity, as did changing the orientation of the nucleophilic tyrosine OH group. Only tyrosine analogues having the phenolic OH group in the normal position with respect to the protein backbone were active; the relative activities could be rationalized in chemical terms on the basis of the H-bonding and the electronic effects of the substituents attached to the meta position of the aromatic ring. In addition, the poisoning of one of the modified human topoisomerase I analogues, as part of covalent binary complexes with DNA, by CPT and 20-thio CPT was evaluated.
    Biochemistry 08/2006; 45(27):8402-10. · 3.38 Impact Factor
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    ABSTRACT: The mechanism of type IB topoisomerase-mediated DNA relaxation was studied by modification of vaccinia topoisomerase I at the active site tyrosine (position 274) with several tyrosine analogues. These analogues had varied steric, electronic, and stereochemical features to permit assessment of those structural elements required to support topoisomerase function. Eleven tyrosine analogues were successfully incorporated into the active site of vaccinia topoisomerase I. It was found that only tyrosine analogues having the phenolic -OH group in the normal position relative to the protein backbone were active. Modifications that replaced the nucleophilic tyrosine OH (pKa approximately 10.0) group with NH2 (pKa 4.6), SH (pKa approximately 7.0), or I groups or that changed the orientation of the nucleophilic OH group essentially eliminated topoisomerase I function. For the active analogues, the electronic effects and H-bonding characteristics of substituents in the meta-position of the aromatic ring may be important in modulating topoisomerase I function. The pH profile for the functional analogues revealed a small shift toward lower pH when compared with wild-type topoisomerase I.
    Journal of the American Chemical Society 04/2005; 127(10):3321-31. · 10.68 Impact Factor