Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

Strategies of organic phosphorus recycling by soil bacteria: acquisition, metabolism, and regulation.

Critical to meeting cellular phosphorus (P) demand, soil bacteria deploy a number of strategies to overcome limitation in inorganic P (Pi ) in soils. As a significant contributor to P recycling, soil bacteria secrete extracellular enzymes to degrade organic P (Po ) in soils into the readily bioavailable Pi . In addition, several Po compounds can be transported directly via specific transporters and subsequently enter intracellular metabolic pathways. In this review, we highlight the strategies that soil bacteria employ to recycle Po from the soil environment. We discuss the diversity of extracellular phosphatases in soils, the selectivity of these enzymes towards various Po biomolecules and the influence of the soil environmental conditions on the enzyme's activities. Moreover, we outline the intracellular metabolic pathways for Po biosynthesis and transporter-assisted Po and Pi uptake at different Pi availabilities. We further highlight the regulatory mechanisms that govern the production of phosphatases, the expression of Po transporters and the key metabolic changes in P metabolism in response to environmental Pi availability. Due to the depletion of natural resources for Pi , we propose future studies needed to leverage bacteria-mediated P recycling from the large pools of Po in soils or organic wastes to benefit agricultural productivity.

Molecular Coordination, Structure, and Stability of Metal-Polyphosphate Complexes Resolved by Molecular Modeling and X-ray Scattering: Structural Insights on the Biological Fate of Polyphosphate.

Polyphosphate-accumulating organisms (PAOs), which can store high levels of phosphate (Pi) in the form of polyphosphate (polyP), are employed to engineer enhanced biological P removal (EBPR) from wastewaters. Co-localization of Mg and K in polyP granules of PAOs has been reported, and higher abundance of Mg-polyP granules relative to other metal complexes was correlated positively with EBPR performance stability. However, the underlying mechanism remains unknown. Here, we obtained molecular structural information of hydrated polyP complexes with four physiologically relevant metal cations (Na+, K+, Ca2+, and Mg2+) using computational and experimental techniques. Molecular dynamics simulations revealed that Mg-polyP and K-polyP complexes were the most and least stable of the complexes, respectively, suggesting that the co-occurrence of these complexes facilitates variable polyP bioavailability. The relative thermodynamic stability reflected the strength of metal chelation whereby the coordination distance between the polyP ligand O and the metal was 1.71-2.01 Å for Mg2+ but this distance was 2.64-2.70 Å for K+. Pair distribution function analysis of X-ray scattering data obtained with a Mg-polyP solution corroborated the theoretical Mg-polyP coordination geometry. These findings implied a possible mechanistic role of metal complexation in the P cycling traits of PAOs in engineered and natural systems.

Hierarchical Reactivity of Enzyme-Mediated Phosphorus Recycling from Organic Mixtures by Aspergillus niger Phytase.

Biological recycling of inorganic phosphorus (Pi) from organic phosphorus (Po) compounds by phosphatase-type enzymes, including phytases, is an important contributor to the pool of bioavailable P to plants and microorganisms. However, studies of mixed-substrate reactions with these enzymes are lacking. Here, we explore 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 Po compounds such as ribonucleotides and sugar phosphates. Using a high-resolution liquid chromatography-mass spectrometry method to monitor simultaneously the parent Po compounds and their by-products, we captured sequential substrate-specific evolution of Pi from the mixture, with faster hydrolysis of multiphosphorylated compounds (phytate, diphosphorylated sugars, and di- and tri-phosphorylated ribonucleotides) than hydrolysis of monophosphorylated compounds (monophosphorylated sugars and monophosphorylated ribonucleotides). The interaction mechanisms and energies revealed by molecular docking simulations of each Po compound within the enzyme's active site explained the substrate hierarchy observed experimentally. Specifically, the favorable orientation for binding of the negatively charged phosphate moieties with respect to the positive potential surface of the active site was important. Collectively, our findings provide mechanistic insights about the broad but hierarchical role of phytase-type enzymes in Pi recycling from the heterogeneous assembly of Po compounds in agricultural soils or wastes.

Abiotic phosphorus recycling from adsorbed ribonucleotides on a ferrihydrite-type mineral: Probing solution and surface species.

Iron (Fe) (oxyhydr)oxide minerals, which are amongst most reactive minerals in soils and sediments, are known to exhibit strong adsorption of inorganic phosphate (Pi) and organophosphate (Po) compounds. Beyond synthetic Po compounds, much still remains unknown about the reactivity of these minerals to transform naturally-occurring Po compounds to Pi, particularly with respect to solution versus surface speciation of Po hydrolysis. To investigate this reactivity with a ferrihydrite-type mineral and ribonucleotides, we employed high-resolution liquid chromatography-mass spectrometry (LC-MS), X-ray absorption near-edge structure (XANES), Fourier-transform infrared (FTIR) spectroscopy, and molecular modeling. Kinetic experiments were conducted with the mineral (1 g L-1) reacted with adenosine monophosphate, diphosphate, or triphosphate (respectively AMP, ADP, ATP; 50 µM). Analysis of solution organic species by LC-MS implied that only adsorption occurred with AMP and ADP but both adsorption and dephosphorylation of ATP were evident. Maximum adsorption capacities per gram of mineral were 40.6 ±â€¯0.8 µmol AMP, 35.7 ±â€¯1.6 µmol ADP, and 10.9 ±â€¯1.0 µmol ATP; solution dephosphorylated by-products accounted for 15% of initial ATP. Subsequent XANES analysis of the surface species revealed that 16% of adsorbed AMP and 30% of adsorbed ATP were subjected to dephosphorylation, which was not fully quantifiable from the solution measurements. Molecular simulations predicted that ADP and ATP were complexed mainly via the phosphate groups whereas AMP binding also involved multiple hydrogen bonds with the adenosine moiety; our FTIR data confirmed these binding confirmations. Our findings thus imply that specific adsorption mechanisms dictate the recycling and subsequent trapping of Pi from ribonucleotide-like biomolecules reacted with Fe (oxyhydr)oxide minerals.

Interactions of oxytetracycline with a smectite clay: a spectroscopic study with molecular simulations.

Binding of antibiotics to clay minerals can decrease both their physical and biological availability in soils. To elucidate the binding mechanisms of tetracycline antibiotics on smectite clays as a function of pH, we probed the interactions of oxytetracycline (OTC) with Na-montmorillonite (MONT) using X-ray diffraction (XRD), infrared (IR), and solid-state nuclear magnetic resonance (NMR) spectroscopies, and Monte Carlo molecular simulations. The XRD patterns demonstrate the presence of OTC in the MONT interlayer space at acidic pH whereas complexation of OTC by external basal and edge sites seems to prevail at pH 8. At both pH, the (1)H-(13)C NMR profile indicates restricted mobility of the adsorbed OTC species; and, -CH(3) deformation and C-N stretching IR vibration bands confirm a binding mechanism involving the protonated dimethylamino group of OTC. Changes in the (23)Na NMR environments are consistent with cation-exchange and cation complexation reactions at the different sites of adsorption. Molecular simulations indicate that MONT interlayer spacing and structural charge localization dictate favorable binding conformations of the intercalated OTC, facilitating multiple interactions in agreement with the spectroscopic data. Our results present complementary insights into the mechanisms of adsorption of TETs on smectites important for their retention in natural and engineered soil environments.

Molecular modeling of metal complexation by a fluoroquinolone antibiotic.

An understanding of the factors controlling the chemodynamics of fluoroquinolone antibiotics in different environmental matrices is a necessary prerequisite to the assessment of their potential impact on nontarget organisms in soils and receiving waters. Of particular interest are the complexes formed between fluoroquinolones and metal cations, which are believed to be important in the mechanism of sequestration of the antibiotic by minerals and natural organic matter. The structures of these complexes have not been fully resolved by conventional spectroscopy; therefore, molecular simulations may provide useful complementary insights. We present results from apparently the first molecular dynamics simulations of a widely used fluoroquinolone antibiotic, ciprofloxacin (Cipro), in aqueous complexes with five metal cations typically found in soils and surface waters: Ca2+, Mg2+, Fe2+, Na+, and K+. The interatomic potential functions employed in the simulations were validated by comparison with available structural data for solid-phase Cipro-hexahydrate and for the metal cations in aqueous solution. Although no comprehensive structural data on the aqueous complexes appear to be available, properties of the metal complexes predicted by our simulations agree with available data for solid-phase metal-Cipro complexes. Our results indicate that the ionic potential of the metal cation controls the stability of the complex formed and that the hydration number of the metal cation in aqueous solution determines its coordination number with O atoms in the metal-Cipro complex. In respect to environmental chemodynamics, our results imply that Cipro will form two configurations of bidendate chelates with metal centers on exposed surfaces of mineral oxides, water-bridged surface complexes with exchangeable cations in clay mineral interlayers, and cation-bridged complexes with functional groups in natural organic matter.