Structural insights into the substrate binding mechanism of novel ArgA from Mycobacterium tuberculosis

Int J Biol Macromol. 2019 Mar 15:125:970-978. doi: 10.1016/j.ijbiomac.2018.12.163. Epub 2018 Dec 18.

Abstract

The Mycobacterium tuberculosis (Mtb) Rv2747 gene encodes for a functional protein known as ArgA, which plays an important role in the first step of the l-arginine biosynthesis pathway. ArgA transfers the acetyl group from the acetyl-CoA to either l-glutamate or l-glutamine, which are the known substrates. Here, we present two crystal structures of ArgA: one complexed with CoA and product bound N-acetylglutamine and the other complexed with acetyl-CoA and the inhibitor l-arginine at 2.3 and 3.0 Å resolution respectively. The Mtb ArgA protomer was found to have a "V" cleft and a "β" bulge, archetypal of a classical GCN5-related N-acetyltransferase superfamily of proteins. The product bound form implies that ArgA can also acetylate l-glutamine like l-glutamate. The active site is strongly inhibited by l-arginine resulting in a closed conformation of ArgA and both l-arginine and N-acetylglutamine were found to occupy at the same active site. Together with structural analysis, molecular docking studies, microscale thermophoresis and enzyme inhibition assays, we conclude that l-glutamine, l-glutamate and l-arginine, all occupy at the same active site of ArgA. Furthermore in case of Mtb ArgA, l-arginine does not act as an allosteric inhibitor unlike other N-acetylglutamate synthase family of proteins.

Keywords: ArgA; Microscale thermophoresis; Mycobacterium tuberculosis; N-acetylglutamine; Rv2747; l-Glutamine.

MeSH terms

  • Acetyl Coenzyme A / chemistry*
  • Acetyl Coenzyme A / metabolism
  • Acetyltransferases / chemistry*
  • Acetyltransferases / genetics
  • Acetyltransferases / metabolism
  • Arginine / chemistry*
  • Arginine / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalytic Domain
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Glutamic Acid / chemistry*
  • Glutamic Acid / metabolism
  • Glutamine / analogs & derivatives
  • Glutamine / chemistry*
  • Glutamine / metabolism
  • Kinetics
  • Molecular Docking Simulation
  • Mycobacterium tuberculosis / chemistry*
  • Mycobacterium tuberculosis / enzymology
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Protein Subunits
  • Recombinant Proteins
  • aceglutamide
  • Glutamine
  • Glutamic Acid
  • Acetyl Coenzyme A
  • Arginine
  • Acetyltransferases