X-ray Crystallography and Electron Paramagnetic Resonance Spectroscopy Reveal Active Site Rearrangement of Cold-Adapted Inorganic Pyrophosphatase.

Scientific Reports
Masaki HoritaniKeiichi Watanabe

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' structu...Continue Reading

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Citations

Nov 17, 2020·Biophysics and Physicobiology·Hitomi Sawai, Koichiro Ishimori
Feb 14, 2021·International Journal of Molecular Sciences·Ilya Lyagin, Elena Efremenko

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Methods Mentioned

BETA
X-ray
gel-filtration
ESR

Software Mentioned

Phaser
XDS )
ARP
EasySpin
wARP
Coot
CCP4
Matlab

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