Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies |
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Authors: | Sagar Chittori Dhirendra Kumar Simanshu Sanchari Banerjee Ambika Mosale Venkatesh Murthy Subashini Mathivanan Handanahal Subbarao Savithri Mathur Ramabhadrashastry Narasimha Murthy |
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Institution: | 1. Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India;2. Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560012, India |
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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 (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. |
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Keywords: | SCFA Short-chain fatty acid TdcD propionate kinase AckA acetate kinase ASKHA Acetate and Sugar Kinase/Heat shock cognate (Hsc) 70/Actin |
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