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

Abstract

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.