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.

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.

Mechanistic modeling of light-induced chemotactic infiltration of bacteria into leaf stomata.

Light is one of the factors that can play a role in bacterial infiltration into leafy greens by keeping stomata open and providing photosynthetic products for microorganisms. We model chemotactic transport of bacteria within a leaf tissue in response to photosynthesis occurring within plant mesophyll. The model includes transport of carbon dioxide, oxygen, bicarbonate, sucrose/glucose, bacteria, and autoinducer-2 within the leaf tissue. Biological processes of carbon fixation in chloroplasts, and respiration in mitochondria of the plant cells, as well as motility, chemotaxis, nutrient consumption and communication in the bacterial community are considered. We show that presence of light is enough to boost bacterial chemotaxis through the stomatal opening and toward photosynthetic products within the leaf tissue. Bacterial chemotactic ability is a major player in infiltration, and plant stomatal defense in closing the stomata as a perception of microbe-associated molecular patterns is an effective way to inhibit the infiltration.

Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida.

Pseudomonas species thrive in different nutritional environments and can catabolize divergent carbon substrates. These capabilities have important implications for the role of these species in natural and engineered carbon processing. However, the metabolic phenotypes enabling Pseudomonas to utilize mixed substrates remain poorly understood. Here, we employed a multi-omics approach involving stable isotope tracers, metabolomics, fluxomics, and proteomics in Pseudomonas putida KT2440 to investigate the constitutive metabolic network that achieves co-utilization of glucose and benzoate, respectively a monomer of carbohydrate polymers and a derivative of lignin monomers. Despite nearly equal consumption of both substrates, metabolite isotopologues revealed nonuniform assimilation throughout the metabolic network. Gluconeogenic flux of benzoate-derived carbons from the tricarboxylic acid cycle did not reach the upper Embden-Meyerhof-Parnas pathway nor the pentose-phosphate pathway. These latter two pathways were populated exclusively by glucose-derived carbons through a cyclic connection with the Entner-Doudoroff pathway. We integrated the 13C-metabolomics data with physiological parameters for quantitative flux analysis, demonstrating that the metabolic segregation of the substrate carbons optimally sustained biosynthetic flux demands and redox balance. Changes in protein abundance partially predicted the metabolic flux changes in cells grown on the glucose:benzoate mixture versus on glucose alone. Notably, flux magnitude and directionality were also maintained by metabolite levels and regulation of phosphorylation of key metabolic enzymes. These findings provide new insights into the metabolic architecture that affords adaptability of P. putida to divergent carbon substrates and highlight regulatory points at different metabolic nodes that may underlie the high nutritional flexibility of Pseudomonas species.

Substrate binding versus escape dynamics in a pH-affected fungal beta-glucosidase revealed by molecular dynamics simulations.

The cellulolytic ability of fungal species is important to both natural and engineered biocycling of plant matter. One essential step is the conversion of cellobiose into glucose catalyzed by beta-glucosidases. Mutagenesis studies have implicated altering the substrate binding pocket to influence the pH-activity profile of this enzyme. However, structural understanding of the pH-affected substrate binding environment is lacking. Here we conducted molecular dynamics simulations of fully hydrated TrBgl2, a beta-glucosidase of Trichoderma reesei, equilibrated at its optimal pH (pH 6) and two unfavorable pHs (pH 5 and pH 7.5). We identified structural arrangement of specific residues that facilitated substrate escape from the catalytic site at pH 5 but locked the bound substrate in an unfavorable orientation at pH 7.5. For comparative analysis, we also performed simulations of a mutated TrBgl2 with previously demonstrated improved catalysis as a function of pH. We captured the responsible conformational changes in the engineered substrate binding pocket.