Enzyme-mediated recycling of inorganic phosphorus (Pi) as orthophosphate from organic P (Porg) compounds is an important contributor to the pool of bioavailable P to plants and microorganisms. Therefore, of special interest is the reactivity of phosphatase-type enzymes of different biological origins (i.e., from plant, fungi, and bacteria) present in agricultural soils towards the soil organic matter enriched a mixture of Porg compounds. This talk will present investigations of the reactivity of a phytase extract from the fungus Aspergillus niger toward a heterogeneous mixture containing, in addition to phytate, different structures of environmentally relevant Porg compounds such as ribonucleotides and sugar phosphates. Using a high-resolution liquid chromatography−mass spectrometry method to monitor simultaneously the parent Porg compounds and their by-products, we captured sequential substrate-specific evolution of Pi from the mixture. Our experimental data captured dephosphorylation activity toward all investigated compounds, but the reactions kinetics were faster (by up to two orders of magnitude) for the multi-phosphorylated compounds (phytate, dephosphorylated sugars, and di- and tri-phosphorylated ribonucleotides) than for the monophosphorylated compounds (monophosphorylated sugars and monophosphorylated ribonucleotides). Our molecular docking simulations revealed that the positively charged potential surface along the enzyme’s active site facilitated favorable binding interactions with multi-phosphorylated compounds, which were consistent with the substrate hierarchy observed experimentally. Compared to single-substrate scenarios, the mixture of competitive substrates resulted in a decrease in the dephosphorylation rates of a favorable substrates (i.e., multi-phosphorylated compounds) but this inhibitory effect was relieved by increasing the concentration of the specific substrate in the mixture. Interestingly, there was no mixture-induced effects for the non-favorable substrates (i.e., monophosphorylated compounds) compared to their single-substrate kinetics, perhaps due to due to persistent low activity for these substrates. The application of high-resolution liquid chromatography-mass spectrometry offers a new approach for probing P recycling activity of enzymes reacted with a mixture of Porg substrates.

Elucidation of Hierarchical Phytase-Catalyzed Phosphorus Recycling from Organic Mixtures using High-Resolution Mass Spectrometry Coupled with Molecular Docking Simulations

ACS Fall 2021

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