Abstract
Inorganic pyrophosphatase (PPase) catalyses the hydrolysis reaction of inorganic pyrophosphate to phosphates. Our previous studies showed that manganese (Mn) activated PPase from the psychrophilic bacterium Shewanella sp. AS-11 (Mn-Sh-PPase) has a characteristic temperature dependence of the activity with an optimum at 5 °C. Here we report the X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy structural analyses of Sh-PPase in the absence and presence of substrate analogues. We successfully determined the crystal structure of Mn-Sh-PPase without substrate and Mg-activated Sh-PPase (Mg-Sh-PPase) complexed with substrate analogue (imidodiphosphate; PNP). Crystallographic studies revealed a bridged water placed at a distance from the di-Mn centre in Mn-Sh-PPase without substrate. The water came closer to the metal centre when PNP bound. EPR analysis of Mn-Sh-PPase without substrate revealed considerably weak exchange coupling, whose magnitude was increased by binding of substrate analogues. The data indicate that the bridged molecule has weak bonds with the di-Mn centre, which suggests a ‘loose’ structure, whereas it comes closer to di-Mn centre by substrate binding, which suggests a ‘well-tuned’ structure for catalysis. Thus, we propose that Sh-PPase can rearrange the active site and that the ‘loose’ structure plays an important role in the cold adaptation mechanism.
Highlights
Structural change of the M2 site is considered the main reason for family II PPases requiring transition metal ions for maximum activity
X-ray crystallography and Electron paramagnetic resonance (EPR) spectroscopy were used for the analysis of the overall and active site structural change of Sh-PPase induced by the substrate
The active site of Mg-Sh-PPase complexed with PNP shows common coordination sphere, like other family II PPases[4,9]
Summary
Structural change of the M2 site is considered the main reason for family II PPases requiring transition metal ions for maximum activity. Some di-Mg2+ and di-Zn2+ enzymes in native form retain their activity when substituted with di-Mn2+ ions, including S-adenosylmethionine synthetase[20], concanavalin A21, enolase[22], ribonucleotide reductase[23,24] and ribonuclease H25 In almost all these enzymes for which crystal structures have been determined, the divalent metals were bridged by a water/hydroxide and one or two carboxylates from aspartate/glutamate residues and the remaining ligands of each metal were coordinated with oxygen residues of carboxylate and/or nitrogen residues of histidine. Such di-Mn2+ centres (two 55Mn, 3d5 high-spin; S1 = S2 = 5/2, I1 = I2 = 5/2) experience a weak antiferromagnetic exchange coupling, which yields total spin S = 0 in the ground state and S = 1, 2, ... The findings reveal the cold adaptation mechanism in Sh-PPase
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