Direct observation of humic acid-promoted hydrolysis of phytate through stabilizing a conserved catalytic domain in phytase

As a potential phosphorus (P) pool, the enzymatic hydrolysis of organic phosphorus (P-o) is of fundamental importance due to the release of bioavailable inorganic phosphate (P-i) for agronomic P sustainability. However, little is known about the role of soil organic matter (SOM) in the hydrolysis process of phytate by phytase and the subsequent chemical behaviors involving the hydrolysis product (P-i) at different soil interfaces. Here, by using liquid-cell atomic force microscopy (AFM), we present a model system to in situ quantify the nucleation kinetics of phytase-released P-i when precipitating with representative soil multivalent cations (Ca2+/Fe3+) on typical soil mineral/organic interfaces in the presence/absence of humic acid (HA), which involves complex phytase-interface-HA interactions. We observed that a higher HA concentration resulted in a faster nucleation rate of amorphous calcium/iron phosphate (ACP/AIP) on bare and organically-coated (-OH/-COOH) mica surfaces compared with the HA-free control. Besides, the nucleation rate of ACP/AIP induced by organic interfaces was much more significant than that induced by clay mineral interfaces. By combining enzyme activity/stability experiments and AFM-based PeakForce quantitative nanomechanical mapping (PF-QNM) measurements, we directly quantified the contribution of noncovalent phytase-HA interaction to the increase in enzymatic activity from complex phytase-interface-HA interactions. Furthermore, the direct complexation of phytase-HA resulted in the stabilization of a conserved active catalytic domain (ACD) in phytase through the enhanced formation of both an ordered, stereochemically-favored catalytic domain and an unordered non-catalytic domain, which was revealed by Raman secondary structure determination. The results provide direct insights into how HA regulates the catalytic activity of phytase controlling P-o fates and how soil interfaces determine the behaviors of released P-i to affect its availability, and thereby contribute to P sustainability in soils.