Use of sequence microdivergence in mycobacterial ortholog to analyze contributions of the water-activating loop histidine of Escherichia coli uracil–DNA glycosylase in reactant binding and catalysis

Acharya, Narottam and Talawar, Ramappa K and Purnapatre, Kedar and Varshney, Umesh (2004) Use of sequence microdivergence in mycobacterial ortholog to analyze contributions of the water-activating loop histidine of Escherichia coli uracil–DNA glycosylase in reactant binding and catalysis. In: Biochemical and Biophysical Research Communications, 320 (3). pp. 893-899.

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Abstract

Uracil–DNA glycosylase (Ung), a DNA repair enzyme, pioneers uracil excision repair pathway. Structural determinations and mutational analyses of the Ung class of proteins have greatly facilitated our understanding of the mechanism of uracil excision from DNA. More recently, a hybrid quantum-mechanical/molecular mechanical analysis revealed that while the histidine (H67 in EcoUng) of the GQDPYH motif ($\omega$ loop) in the active site pocket is important in positioning the reactants, it makes an unfavorable energetic contribution (penalty) in achieving the transition state intermediate. Mutational analysis of this histidine is unavailable from any of the Ung class of proteins. A complication in demonstrating negative role of a residue, especially when located within the active site pocket, is that the mutants with enhanced activity are rarely obtained. Interestingly, unlike the most Ung proteins, the H67 equivalent in the $\omega$ loop in mycobacterial Ung is represented by P67. Exploiting this natural diversity to maintain structural integrity of the active site, we transplanted an H67P mutation in EcoUng. Uracil inhibition assays and binding of a proteinaceous inhibitor, Ugi (a transition state substrate mimic), with the mutant (H67P) revealed that its active site pocket was not perturbed. The catalytic efficiency $(V_{max}/K_m)$ of the mutant was similar to that of the wild type Ung. However, the mutant showed increased $K_m$ and $V_{max.}$ Together with the data from a double mutation H67P/G68T, these observations provide the first biochemical evidence for the proposed diverse roles of H67 in catalysis by Ung.

Item Type:	Journal Article
Publication:	Biochemical and Biophysical Research Communications
Publisher:	Elsevier Science
Additional Information:	Copyright of this article belongs to Elsevier Science.
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	22 Nov 2006
Last Modified:	23 Feb 2012 10:15
URI:	http://eprints.iisc.ac.in/id/eprint/6458

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