Fig 1.
The HMG/CHA aldolase reactions and active site.
(A) Aldol cleavage catalyzed by the HMG/CHA aldolase where for HMG the R group is -CH3 which yields 2 moles of pyruvate and for CHA the R group is -CH2COO- yielding 1 mole each of pyruvate and oxaloacetate. (B) Oxaloacetate decarboxylase reaction catalyzed by the aldolase producing either carbon dioxide or bicarbonate. (C) Active site of the aldolase showing the pyruvate carbon in yellow sticks, the bound magnesium ion as a green sphere, and key water molecules as red spheres. The structural representation was generated in Pymol (version 1.7) using atomic coordinates of the HMG/CHA aldolase (PDB ID: 3NOJ).
Table 1.
Pi activation with different substrates.
Table 2.
Pyruvate methyl proton exchange rate of the wild type and variants of the HMG/CHA aldolase in the absence and presence of Pi.
Fig 2.
Docking of Pi into the active site of the HMG/CHA aldolase.
The HMG/CHA aldolase as represented in Fig 1. (A) The center of mass of each Pi ion is shown as an orange sphere and the grouped putative binding sites are numbered. A representative binding mode for sites 1 (B), 2 (C), and 3 (D) are shown with the native structures residues as lines with significant positional changes indicated with arrows. Potential hydrogen bonds between Pi and the protein, defined by distances between 2.8 and 3.2 Å, are indicated by dashes.
Table 3.
Steady state kinetic parameters of HMG lyase activity of variants of the HMG/CHA aldolase in the presence or absence of the Pi.
Fig 3.
Comparison of the HMG/CHA aldolase with the PT proteins.
(A) Overall structural comparison with the proteins oriented via structural alignment of the conserved domain which is coloured white with arrows pointing out the aldolase Arg-123 and transferase PH residue. The HMG/CHA aldolases Mg2+ and pyruvate are shown as a green sphere and yellow sticks, respectively. EI:PTS from E. coli (PDB: 2HWG) is shown with its PEP binding domain coloured red its and helical bundle, which supports HPr binding, coloured green. The EI:PTS Mg2+ and oxalate are shown as a green sphere and yellow sticks, respectively. PK from Geobacillus strearothermophilus (PDB: 2E28) is shown with its kinase domain coloured magenta its and effector domain coloured dark green. The PPDK from Maize (PDB: 1VBH) is shown with its nucleotide binding domain, linker region, and PEP binding domain coloured in cyan, pink, and brown, respectively. The PPDK Mg2+ is shown as a green sphere and its bound PEP as yellow sticks. (B) Section of the primary sequences in the structural alignment indicating the HMG/CHA aldolases essential Arg-123 in a magenta box, the PH with an orange sphere, and the PPDK threonine observed as a site of phosphorylation in plants as a green sphere. (C) Overlay of the HMG/CHA aldolase and EI:PTS with comparison of key residues. The enzymes are coloured as in panel A except, for clarity, the conserved domain in EI:PTS is coloured blue.