Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism

Murthy, Ambika Mosale Venkatesh, Mathivanan, Subashini, Chittori, Sagar, Savithri, Handanahal Subbarao and Murthy, Mathur Ramabhadrashastry Narasimha (2015) Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism. Acta Crystallographica Section D: Biological Crystallography, 71 1640-1648. doi:10.1107/S1399004715009992

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Author Murthy, Ambika Mosale Venkatesh
Mathivanan, Subashini
Chittori, Sagar
Savithri, Handanahal Subbarao
Murthy, Mathur Ramabhadrashastry Narasimha
Title Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism
Formatted title
Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism
Journal name Acta Crystallographica Section D: Biological Crystallography   Check publisher's open access policy
ISSN 1399-0047
Publication date 2015-08-01
Sub-type Article (original research)
DOI 10.1107/S1399004715009992
Open Access Status File (Publisher version)
Volume 71
Start page 1640
End page 1648
Total pages 9
Place of publication Malden, MA, United States
Publisher Wiley-Blackwell Publishing
Collection year 2016
Language eng
Formatted abstract
Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of L-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 Å resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of ~5 Å from the γ-phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative SN2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the β- and the γ- phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the γ-phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity.
Keyword Direct in-line phosphate transfer
Kinetic characterization
SCFA metabolism
Site-directed mutagenesis
StTdcD
Substrate specificity
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
Institute for Molecular Bioscience - Publications
 
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