کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8331748 | 1540245 | 2015 | 29 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
AMP-acetyl CoA synthetase from Leishmania donovani: Identification and functional analysis of 'PX4GK' motif
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موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A â¼77Â kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5â²-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: International Journal of Biological Macromolecules - Volume 75, April 2015, Pages 364-372
Journal: International Journal of Biological Macromolecules - Volume 75, April 2015, Pages 364-372
نویسندگان
Neelagiri Soumya, I. Sravan Kumar, S. Shivaprasad, Landage Nitin Gorakh, Neeradi Dinesh, Kayala Kambagiri Swamy, Sushma Singh,