title: Genetically engineered synthetic miniaturized versions of Plasmodium falciparum UvrD helicase are catalytically active.
abstract: Helicases catalyze unwinding of double stranded nucleic acids in an energy-dependent manner. We have reported characterization of UvrD helicase from Plasmodium falciparum. We reported that the N-terminal and C-terminal fragments of PfUvrD contain characteristic ATPase and DNA helicase activities. Here we report the generation and characterization of a genetically engineered version of PfUvrD and its derivatives. This synthetic UvrD (sUD) contains all the conserved domains of PfUvrD but only the intervening linker sequences are shortened. sUD (∼ 45 kDa) and one of its smallest derivative sUDN1N2 (∼ 22 kDa) contain ATPase and DNA helicase activities. sUD and sUDN1N2 can utilize hydrolysis of all the NTPs and dNTPs, can also unwind blunt end duplex DNA substrate and unwind DNA duplex in 3 to 5 direction only. Some of the properties of sUD are similar to the PfUvrD helicase. Mutagenesis in the conserved motif Ia indicate that the mutants sUDM and sUDN1N2M lose all the enzyme activities, which further confirms that these activities are intrinsic to the synthesized proteins. These studies show that for helicase activity only the conserved domains are essentially required and intervening sequences have almost no role. These observations will aid in understanding the unwinding mechanism by a helicase.
authors.affiliation: Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Abulaish
authors.initials: A
authors.lastname: Ansari
authors.affiliation: Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Tarique
authors.affiliation: Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Renu
authors.initials: R
authors.lastname: Tuteja
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000201:enzymology; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases