Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

• Published in: Biochimica et biophysica acta 2013-10, Vol.1834 (10), p.2036-2044
• Main Authors: Chittori, Sagar, Simanshu, Dhirendra Kumar, Banerjee, Sanchari, Murthy, Ambika Mosale Venkatesh, Mathivanan, Subashini, Savithri, Handanahal Subbarao, Murthy, Mathur Ramabhadrashastry Narasimha
• Format: Article
• Language: English
• Subjects: adenosine; adenosine monophosphate; adenosine triphosphate; Alanine - chemistry; Alanine - genetics; alpha-ketoglutaric acid; Amino Acid Sequence; bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; carbon; carbon cycle; catalytic activity; citrates; crystal structure; Crystallography, X-Ray; dimerization; energy; Enzyme assay; Enzyme Assays; ethylene glycol; Ethylene Glycol - chemistry; guanosine triphosphate; Hydrophobic and Hydrophilic Interactions; hydrophobic bonding; Kinetics; Magnesium - chemistry; malates; Manganese - chemistry; metal ions; molecular dynamics; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleotides - chemistry; Phosphotransferases (Carboxyl Group Acceptor) - chemistry; Phosphotransferases (Carboxyl Group Acceptor) - genetics; Propionates - chemistry; propionic acid; Protein dynamics; Protein Multimerization; Salmonella Typhimurium; Salmonella typhimurium - chemistry; Salmonella typhimurium - enzymology; Sequence Alignment; short chain fatty acids; Short-chain fatty acid metabolism; site-directed mutagenesis; Substrate Specificity; succinic acid; threonine; tricarboxylic acid cycle; X-ray crystallography
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2013.05.020

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 (Mg2+≈Mn2+>Co2+). 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. •Biochemical specificity and regulatory properties of StTdcD were studied.•Purine and pyrimidine nucleotide bound structures of StTdcD were determined.•Molecular basis for broad nucleotide and substrate specificity was examined.•Molecular dynamics simulations provided insights on domain motion upon ligand binding.•Analysis of StTdcD complexes supports direct in-line phosphoryl transfer mechanism.