title: Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): insights from kinetic and crystallographic studies.
abstract: Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the γ-phosphate of ATP to propionate during l-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate>acetate≈butyrate), nucleotides (ATP≈GTP>CTP≈TTP; dATP>dGTP>dCTP) and metal ions (Mg(2+)≈Mn(2+)>Co(2+)). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, α-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO4 (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235-290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the γ-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India. Electronic address: sagar@mbu.iisc.ernet.in.
authors.forename: Sagar
authors.initials: S
authors.lastname: Chittori
authors.forename: Dhirendra Kumar
authors.initials: DK
authors.lastname: Simanshu
authors.forename: Sanchari
authors.initials: S
authors.lastname: Banerjee
authors.forename: Ambika Mosale Venkatesh
authors.initials: AM
authors.lastname: Murthy
authors.forename: Subashini
authors.initials: S
authors.lastname: Mathivanan
authors.forename: Handanahal Subbarao
authors.initials: HS
authors.lastname: Savithri
authors.forename: Mathur Ramabhadrashastry Narasimha
authors.initials: MR
authors.lastname: Murthy
mesh_terms: D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D057075:Enzyme Assays; D019855:Ethylene Glycol / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry*; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D011422:Propionates / Q000737:chemistry*; D055503:Protein Multimerization; D012486:Salmonella typhimurium / Q000737:chemistry* / Q000201:enzymology; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D011422:Propionates; D008345:Manganese; D017851:Phosphotransferases (Carboxyl Group Acceptor); D019855:Ethylene Glycol; D008274:Magnesium; D000409:Alanine
keywords: ASKHA; Acetate and Sugar Kinase/Heat shock cognate (Hsc) 70/Actin; AckA; Enzyme assay; Protein dynamics; SCFA; Salmonella typhimurium; Short-chain fatty acid; Short-chain fatty acid metabolism; TdcD; X-ray crystallography; acetate kinase; propionate kinase