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bioRxiv · 10.1101/2025.06.08.658471

Formation of a μ-oxo nucleophile enables efficient hydrolysis by a trinuclear metal center in Family II inorganic pyrophosphatase

Maruoka, S.·Kametani, Y.·Magome, E.·Setoyama, H.·Kawamoto, M.·Horitani, M.·Teramoto, T.·Kakuta, Y.·Shiota, Y.·Yoshizawa, K.·Watanabe, K.
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Abstract

Efficient catalysis by metalloproteins relies on precise spatial arrangement of metal ions and active-site residues. Family II inorganic pyrophosphatase (PPase) from Shewanella species features a trinuclear metal center and displays higher catalytic activity than binuclear counterparts. Here we elucidate its hydrolytic mechanism using X-ray crystal structure-based extended X-ray absorption fine structure (XCS-EXAFS), site-directed mutagenesis, and density functional theory (DFT) calculations. We identify a catalytic -oxo nucleophile, formed via proton transfer from a bridging -hydroxide to Asp14 and subsequent hydrogen-bond rearrangement to Asp72, as the key species in SN2-type hydrolysis. This conversion defines the rate-limiting step with an activation barrier of 15.5 kcal/mol. Molecular orbital analysis reveals that the trinuclear cluster promotes -oxo formation, aligns the nucleophile for attack, and stabilizes the transition state. The side-chain rotation of the conserved Asp14 is crucial for catalysis. Our results highlight how metalloenzymes exploit geometric and electronic tuning to achieve high reactivity through evolutionarily optimized architectures.

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Maruoka, S., Kametani, Y., Magome, E., Setoyama, H., Kawamoto, M., Horitani, M., Teramoto, T., Kakuta, Y., Shiota, Y., Yoshizawa, K., Watanabe, K.. 2025-06-09. Formation of a μ-oxo nucleophile enables efficient hydrolysis by a trinuclear metal center in Family II inorganic pyrophosphatase. https://doi.org/10.1101/2025.06.08.658471

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