title: AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.
abstract: 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 ∼ 77 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 5'-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.
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors: Neelagiri
authors: N
authors: Soumya
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors: I Sravan
authors: IS
authors: Kumar
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors: S
authors: S
authors: Shivaprasad
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors: Landage Nitin
authors: LN
authors: Gorakh
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors: Neeradi
authors: N
authors: Dinesh
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors: Kayala Kambagiri
authors: KK
authors: Swamy
authors: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India. Electronic address: sushmasingh@niper.ac.in.
authors: Sushma
authors: S
authors: Singh
mesh_terms: D000106:Acetate-CoA Ligase / Q000737:chemistry* / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D055592:Biophysical Phenomena / Q000187:drug effects; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: D007477:Ions; D008670:Metals; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D000106:Acetate-CoA Ligase
keywords: AMP; Acetate; Acetyl CoA synthetase; Leishmania donovani; PX(4)GK motif