title: Leucine-684: A conserved residue of an AMP-acetyl CoA synthetase (AceCS) from Leishmania donovani is involved in substrate recognition, catalysis and acetylation.
abstract: AMP-acetyl CoA synthetase (AMP-AceCS) is a key enzyme which catalyzes the activation of acetate to acetyl CoA, an important intermediate at the cross roads of various anabolic and catabolic pathways. Multiple sequence alignment of Leishmania donovani AceCS with other organisms revealed the presence of a highly conserved leucine residue at 684 position which is known to be crucial for acetylation by protein acetyl transferases in other organisms. In an attempt to understand the role of leucine residue at 684 position in L. donovani acetyl CoA synthetase (LdAceCS), it was mutated to proline (P) by site directed mutagenesis. Kinetic analysis of the L684P-LdAceCS mutant revealed approximately two fold increased binding affinity with acetate, whereas fivefold decreased affinity was observed with ATP. There was insignificant change in secondary structure as revealed by CD however, two fold decreased fluorescence intensity was observed at an emission maxima of 340 nm. Interestingly, L684P mutation abolished the acetylation of the mutant enzyme indicating the importance of L684 in acetylation of the enzyme. Changes in biochemical parameters of the mutant protein were validated by homology modeling of the wild type and mutant LdAceCS enzyme using Salmonella enterica AceCS crystal structure as template. Our data provides evidence for the role of leucine 684 residue in substrate recognition, catalysis and acetylation of the AceCS enzyme.
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Neelagiri
authors.initials: N
authors.lastname: Soumya
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Hitendra
authors.initials: H
authors.lastname: Tandan
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Mangesh V
authors.initials: MV
authors.lastname: Damre
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Rahul P
authors.initials: RP
authors.lastname: Gangwal
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Abhay T
authors.initials: AT
authors.lastname: Sangamwar
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India. Electronic address: sushmasingh@niper.ac.in.
authors.forename: Sushma
authors.initials: S
authors.lastname: Singh
mesh_terms: D000106:Acetate-CoA Ligase / Q000235:genetics* / Q000378:metabolism*; D000107:Acetylation; D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D017124:Conserved Sequence; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D007930:Leucine / Q000235:genetics*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D000249:Adenosine Monophosphate; D000106:Acetate-CoA Ligase; D007930:Leucine
keywords: AceCS; Homology modeling; Kinetics; L684 mutation; Site directed mutagenesis