Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap₄A: evidence for a novel Ap₄A synthetic activity

Abstract

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'''-P¹,P⁴-tetraphosphate (Ap₄A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap₄A bound to the active site pocket suggesting the presence of Ap₄A synthetic activity in TdcD. Binding of Ap₄A to the enzyme was confirmed by the structure determination of a TdcD-Ap₄A complex obtained after cocrystallization of TdcD with commercially available Ap₄A. Mass spectroscopic studies provided further evidence for the formation of Ap₄A by propionate kinase in the presence of ATP. In the TdcD-Ap₄A complex structure, Ap₄A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.