doi:10.1016/j.jmb.2005.07.069 
Copyright © 2005 Elsevier Ltd All rights reserved.
Crystal Structures of ADP and AMPPNP-bound Propionate Kinase (TdcD) from Salmonella typhimurium: Comparison with Members of Acetate and Sugar Kinase/Heat Shock Cognate 70/Actin Superfamily
Dhirendra K. Simanshua, H.S. Savithrib and M.R.N. Murthya, 
, 
aMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
bDepartment of Biochemistry, Indian Institute of Science, Bangalore 560012, India
Received 1 June 2005;
revised 27 July 2005;
accepted 28 July 2005.
Edited by M. Guss.
Available online 11 August 2005.
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Recently, it has been shown that l-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2 Å and 2.3 Å, respectively, by molecular replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. This was further confirmed by kinetic studies with the purified enzyme, which showed about ten times lower Km for propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well-characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure.
Keywords: propionate kinase; Salmonella typhimurium; TdcD; crystal structure; l-threonine metabolism
Abbreviations used: AMPPNP, adenylyl imidodiphosphate; ATPγS, adenosine 5′-[γ-thio] phosphate; ASKHA, acetate and sugar kinase-heat shock cognate 70-actin; TdcD, propionate kinase from Salmonella typhimurium; MAK, acetate kinase from Methanosarcina thermophila
Figure 1. Amino acid sequence alignment of propionate kinase and acetate kinase from S. typhimurium and M. thermophila. The residues conserved in all four molecules are shown in black boxes. TdcD_ST and PduW_ST denote the propionate kinase encoded by TdcD and PduW in S. typhimurium, whereas AK_ST and AK_MT denote acetate kinase present in S. typhimurium and M. thermophila, respectively. The secondary structure elements of ADP bound TdcD are represented at the top of the aligned sequences by arrows (β-strands) and spirals (310 and α-helices). The red triangles below the aligned sequences show the probable residues involved in catalytic mechanism or substrate binding. The Figure was prepared using CLUSTALW47 and ESPript.48
Figure 2. Crystal structure of propionate kinase. (a) Three-dimensional structure of the homodimeric propionate kinase in complex with ADP, with each subunit coloured differently. The molecular 2-fold axis is along the plane of the paper. (b) Ribbon diagram of a subunit of propionate kinase bound with AMPPNP and ethylene glycol. The secondary structure elements in the subunit are coloured differently. (c) The surface representation of the TdcD-AMPPNP showing the binding site of AMPPNP and ethylene glycol near the proposed binding site of propionate. Positively charged residues are shown in blue, negatively charged residues are shown in red and neutral residues are shown in white. The ligands AMPPNP, ADP and ethylene glycol present in the active site cleft are drawn as ball-and-stick models. (a) and (b) were prepared using MOLSCRIPT43 and Raster3D;45 (c) was prepared using GRASP†.
Figure 3. Topology diagram of the secondary structural elements of TdcD. Arrows represent β-strands, while small and large cylinders represent 310 and α-helices, respectively. β-Strands and α-helices, coloured cyan and green, respectively, constitute the core secondary structure βββαβαβα in both the domains, while insertions of sub-domains between particular secondary structural elements are coloured yellow. The core secondary structure elements βββαβαβα are numbered as β1, β2, β3, α1, β4, α2, β5, α3 in domain I and as β1*, β2*, β3*, α1*, β4*, α2*, β5*, α3* in domain II. Secondary structural elements present in the inserted subdomains are numbered as βa, βb and βc for β-strands, αa, αb and αc for α-helices in domain I, and βa* for β-strand and αa*, αb*, αc*, αd*, αe* and αf* for α-helices in domain II. The 310 helices are numbered as 3a, 3b 3c, 3d, 3e and 3f. The Figure was generated using TOPDRAW.49
Figure 4. Electrostatic potential surface representation illustrating the charge distribution in TdcD at the dimeric interface. The Figure was generated using GRASP†.
Figure 5. (a) Stereo view of the electron density corresponding to AMPPNP in AMPPNP–TdcD complex structure from a 2Fo−Fc map contoured at 1.0σ. (b) Stereo diagram of the active site region showing bound AMPPNP (labelled as ANP) in the TdcD–AMPPNP complex. AMPPNP is shown in ball-and-stick. Hydrogen bonds formed by AMPPNP with protein atoms and water molecules are shown as broken lines. The Figure was prepared using the programs BOBSCRIPT44 and MOLSCRIPT,43 and rendered using Raster3D.45
Figure 6. (a) Stereo view of the electron density corresponding to ADP in the TdcD–ADP complex structure from a 2Fo−Fc map countered at 1.0σ. (b) Stereo diagram of the active site region showing ADP bound to TdcD in the TdcD–ADP complex structure. ADP is shown in ball-and-stick. Hydrogen bonds formed by ADP with protein atoms and water molecules are shown as broken lines. The Figure was prepared using the programs BOBSCRIPT44 and MOLSCRIPT,43 and rendered using Raster3D.45
Figure 7. Stereo diagram of the active site showing ethylene glycol (labelled as EDO) bound near the proposed binding site for propionate in TdcD. The Figure shows the electron density corresponding to ethylene glycol and a water molecule from a 2Fo−Fc map countered at 1.0σ. Ethylene glycol is shown in ball-and-stick. Hydrogen bonds between EDO and nearby atoms are shown as broken lines. This Figure was prepared using the program BOBSCRIPT.44
Figure 8. Alignments of the five putative motifs based on structural equivalence in the well-characterized members of the ASKHA superfamily. Residues shown in bold in each motif correspond to either the conserved catalytic residues or those that have a structurally equivalent role in binding nucleotide or metal. TdcD, propionate kinase from S. typhimurium; AK, acetate kinase from M. thermophila; HK, hexokinase from yeast; GK, glycerol kinase from E. coli; actin, actin from rabbit skeletal muscle; Hsc70, heat shock cognate 70 from Bos taurus.
Figure 9. (a) Interdomain rotation axis within the A and B subunits of the MAK–ATP complex structure determined by the program DynDom.35 Arrow indicates the direction of rotation of the moving domain by the right-hand rule. (b) Structural superposition of TdcD and the A and B subunits of the MAK–ATP structure, showing the movement of domain I (moving domain) relative to the fixed domain (domain II). (c) Structural superposition of the moving domain and hinge residues in TdcD and the A and B subunits of the MAK–ATP structure, indicating that the domain motion is a result of rigid body movement across hinge residues (shown in red). TdcD and the A and B subunits of the MAK–ATP structure are shown in purple, yellow and green, respectively. The Figure was generated using the programs RASMOL42 and MOLSCRIPT,43 and rendered with Raster3D.45
Table 1.
Crystal parameters and statistics on data collection, refinement and model quality
Values in parentheses correspond to the highest resolution shell.
a Rmerge=Σ|Ij−
Ij
|/ΣIj, where
Ij is the intensity of reflection
j and its symmetry equivalents, and
Ij is the average intensity of the reflections.
Table 2.
Residue range in the fixed and the moving domains and in the hinge region of TdcD and MAK
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