Functional Characteristics of Inorganic Pyrophosphatase from Psychrotroph Shewanella sp. AS-11 upon Activation by Various Divalent Cations

Ginting, Elvy Like; Maeganeku, Chihiro; Motoshima, Hidemasa; Watanabe, Kenji

doi:10.14233/ajchem.2014.16217

Cited by 1 publication

(1 citation statement)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the crystals obtained did not show any electron density for PNP, likely because Mn-Sh-PPase only weakly degrades PNP, even in the presence of sodium fluoride. In contrast, Sh-PPase activated by Mg 2+ ions (Mg-Sh-PPase) has much lower catalytic activity with PPi or PNP than Mn-Sh-PPase 13 , and we obtained well-diffracted crystals of Mg-Sh-PPase in the presence of PNP and sodium fluoride. X-ray diffraction data at 1.3 Å resolution were obtained.…”

Section: Resultsmentioning

confidence: 91%

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

Horitani

Kusubayashi

Oshima

et al. 2020

Sci Rep

View full text Add to dashboard Cite

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-Shppase 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 Shppase can rearrange the active site and that the 'loose' structure plays an important role in the cold adaptation mechanism.Inorganic pyrophosphatase (PPase) catalyses the hydrolysis reaction of inorganic pyrophosphate (PPi) to two inorganic phosphates (Pi) and plays an essential role for living organisms 1 . Soluble PPase consists of two families, I and II, which have different secondary and tertiary structures. Family I PPases are widespread organisms including bacteria and mammalians 1 , whereas family II PPases are almost exclusively found in bacteria and archaebacteria 2-4 . Both the family I and II PPases require divalent metal ions for their reactivity. All PPases are magnesium ion (Mg 2+ ) dependent enzymes. Family II PPases require divalent transition metals, such as Mn 2+ 3,5 or Co 2+ ions 6 for maximum activity. X-ray structural analyses of family II PPases from Bacillus subtilis (Bs-PPase) and Streptococcus gordonii (Sg-PPase) have been reported 7,8 . They are homodimers and each monomer consists of two domains (N-and C-terminal domains) connected by a flexible hinge region. These two domains are in the 'open' state in the absence of substrate. Substrate binding results in a 'closed' state, with one exception 7 . Two sites (M1 and M2) for transition metal ion have been demonstrated in the active site in the 'open' state of Bs-PPase, while four metal ions (M1 to M4) are found in the crystal structure of Bs-PPase in complex with imidodiphosphate (PNP) 9 . Metal requirement studies for Bs-PPase have shown that the M2 site is preferred for divalent transition metal ions, such as Mn 2+ or Co 2+ , while other metal binding sites (M3 and M4) favour Mg 2+ ions in catalysis. However, it is still unclear whether the M1 site utilizes a transition m...

show abstract

Section: Resultsmentioning

confidence: 91%