Enzymatic Mechanism Research Articles

Deep quantification of substrate turnover defines protease subsite cooperativity.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.

Supramolecular Catalyst for Improving the Sensitivity of Biomolecule Detection

AbstractSupermolecule refers to two or more molecules that are bonded together by secondary bonds between molecules to form a multimolecular system at the level of more than molecules. At this point, the molecules form an orderly aggregation, showing its unique structure and function. Supramolecular catalysts can form structures with specific functions through non‐covalent interactions (e. g., hydrogen bonding, π‐π stacking, etc.) to mimic the catalytic mechanism of natural enzymes and achieve susceptible and specific detection of biomolecules. The application of supramolecules is mainly focused on the self‐assembly of components for the construction of sensing platform. Therefore, supramolecular catalysts have a huge potential in biomolecule detection. This article focuses on the application of cyclodextrins(CD), cucurbit[n]uril(CB), porphyrins(PP) and Calix[n]arenes(CA) in the field of catalysis and in improving detection sensitivity. Research progress of supramolecular catalysts in polymerization amplification is summarized in this paper.

Cover Feature: Tutorial Review on the Set‐Up and Running of Quantum Mechanical Cluster Models for Enzymatic Reaction Mechanisms (Chem. Eur. J. 60/2024)

Cover Feature: Tutorial Review on the Set‐Up and Running of Quantum Mechanical Cluster Models for Enzymatic Reaction Mechanisms (Chem. Eur. J. 60/2024)

Quantum mechanics/molecular mechanics (QM/MM) methods in drug design: a comprehensive review of development and applications

The integration of quantum mechanics (QM) and molecular mechanics (MM) methods, known as QM/MM, has emerged as a powerful computational approach in drug design. This hybrid technique combines the accuracy of QM calculations for the reactive region with the computational efficiency of MM methods for the surrounding environment. QM/MM methods have proven invaluable in studying chemical reactions, exploring enzyme mechanisms, and investigating ligand-protein interactions, all of which are crucial for rational drug design. This review provides a comprehensive overview of the QM/MM methodology, its theoretical foundations, and its applications in various aspects of drug design. We discussed the key components, such as QM and MM region partitioning, link atom schemes, and boundary treatments. Additionally, we highlight recent advancements and challenges in QM/MM methods, including polarizable force fields, implicit solvation models, and enhanced sampling techniques. Furthermore, we present illustrative examples showcasing the successful application of QM/MM methods in lead optimization, virtual screening, and the elucidation of biochemical mechanisms relevant to drug targets. Finally, we provide perspectives on future developments and the potential impact of QM/MM methods on the drug discovery pipeline.

Antiviral role of cholesterol 25-hydroxylase in inhibiting porcine circovirus 3 replication

Cholesterol 25-hydroxylase (CH25H) has significant antiviral effects through the production of 25-hydroxycholesterol (25HC). In this study, we investigated the effects of CH25H, its catalytic product 25HC, and its catalytic mutant lacking hydroxylase activity (CH25H-M) on porcine circovirus 3 (PCV3) replication. By transfecting PCV3 persistently infected PK-15 cells with the pCAGGS-CH25H-Flag plasmid, the results demonstrated that overexpression of CH25H significantly inhibited PCV3 Cap protein expression, Cap mRNA levels, and viral titers in a dose-dependent manner. Moreover, its catalytic product 25HC inhibited PCV3 replication in PK-15 cells at concentrations below 10 µM without affecting cell viability. In contrast, knockdown of endogenous CH25H using small interfering RNA (siRNA) enhanced PCV3 replication, further confirming its antiviral role. Interestingly, the CH25H-M mutant also exhibited inhibitory effects on PCV3 replication, although the inhibition was much less effective compared with CH25H. In conclusion, CH25H plays a critical role in regulating PCV3 replication, and its antiviral effect is not entirely dependent on its enzymatic activity. These findings provide new insights into both the enzymatic and non-enzymatic antiviral mechanisms of CH25H and revealed some mechanistic immune evasion for PCV3.

Position-specific kinetic isotope effects for nitrous oxide: a new expansion of the Rayleigh model

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and the most significant anthropogenic ozone-depleting substance currently being emitted. A major source of anthropogenic N2O emissions is the microbial conversion of fixed nitrogen species from fertilizers in agricultural soils. Thus, understanding the enzymatic mechanisms by which microbes produce N2O has environmental significance. Measurement of the 15N / 14N isotope ratios of N2O produced by purified enzymes or axenic microbial cultures is a promising technique for studying N2O biosynthesis. Typically, N2O-producing enzymes combine nitrogen atoms from two identical substrate molecules (NO or NH2OH). Position-specific isotope analysis of the central (Nα) and outer (Nβ) nitrogen atoms in N2O enables the determination of the individual kinetic isotope effects (KIEs) for Nα and Nβ, providing mechanistic insight into the incorporation of each nitrogen atom. Previously, position-specific KIEs (and fractionation factors) were quantified using the Rayleigh distillation equation, i.e., via linear regression of δ15Nα or δ15Nβ against [-fln⁡f/(1-f)], where f is the fraction of substrate remaining in a closed system. This approach, however, is inaccurate for Nα and Nβ because it does not account for fractionation at Nα affecting the isotopic composition of substrate available for incorporation into the β position (and vice versa). Therefore, we developed a new expansion of the Rayleigh model that includes specific terms for fractionation at the individual N2O nitrogen atoms. By applying this Expanded Rayleigh model to a variety of simulated N2O synthesis reactions with different combinations of normal, inverse, and/or no KIEs at Nα and Nβ, we demonstrate that our new model is both accurate and robust. We also applied this new model to two previously published datasets describing N2O production from NH2OH oxidation in a methanotroph culture (Methylosinus trichosporium) and N2O production from NO by a purified Histoplasma capsulatum (fungal) P450 NOR, demonstrating that the Expanded Rayleigh model is a useful tool in calculating position-specific fractionation for N2O synthesis.

Microbial depolymerization of Kraft lignin for production of Vanillic acid by indigenous ligninolytic strains

Lignin is a potential renewable source for the production of many value-added bio-products through bacterial depolymerization. In this study, Kraft lignin (KL) was depolymerized by indigenous potential ligninolytic strains to obtain the optimum amount of Vanillic acid. Initially, fifteen bacterial strains were isolated from decaying wood mixed soil to assess their lignin degradation efficiency. Five potential lignin-degrading microbes (RW-B2, RW-B4, RW-B9, RW-B11 and RW-B15) were identified by nutrient enrichment technique. RW-B15 was observed as a dominant strain sharing a 99 % resemblance to Bacillus megaterium sp. Optimum temperature and bacterial inoculum concentration were noted as 35 °C and 1 %, respectively. Mixed microbial culture of five potential ligninolytic strains showed better KL degradation than pure culture due to different metabolic capacities, enzymatic mechanisms and synergetic impact among the association members. The KL degradation study was conducted with different KL concentrations (500–2000 mg/l) at optimum temperature using 1 % inoculums. The production of Vanillic acid increased from 60.1 to 199.9 mg/l with increasing concentration of KL from 500 to 2000 mg/l. The maximum production of Vanillic acid was obtained on 3rd day of degradation and it decreased thereafter which is probably due to an increase in microbial toxicity and enzymatic activity. However, the COD level continuously decreased for all KL concentrations with degradation time till the last day. The pH of the KL solution slightly decreased on 3rd day due to the acidic nature of Vanillic acid. GC-MS analysis confirmed the presence of Vanillic acid and showed the presence of other monomeric compounds formed after KL degradation.

A Phenylalanine Sensor Exploiting a Capacitive Readout Strategy Embedding a Selective Enzymatic Mechanism

A Phenylalanine Sensor Exploiting a Capacitive Readout Strategy Embedding a Selective Enzymatic Mechanism

Insecticide resistance status and mechanisms in Aedes aegypti and Aedes albopictus from different dengue endemic regions of Panama

BackgroundDengue is a serious public health problem worldwide, including Panama. During the last years, the number of dengue cases has increased. This may be due to the presence of mosquito populations resistant to insecticides. The aim of this study was to characterize the resistance status, its enzymatic mechanisms and Kdr mutations in wild populations of Aedes aegypti and Aedes albopictus.MethodsStandard WHO bioassays were performed using insecticide-treated filter papers to determine resistance in populations Ae. aegypti and Ae. albopictus to pyrethroids insecticides, organophosphates, to the carbamate propoxur and to the organochlorine DDT. Biochemical assays were conducted to detect metabolic resistance mechanisms and real-time PCR was performed to determine the frequencies of the Kdr mutations Val1016IIe and F1534C.ResultsThe strains Ae. aegypti El Coco showed confirmed resistance to deltamethrin (78.5% mortality) and lambda-cyhalothrin (81%), Aguadulce to deltamethrin (79.3%), David to deltamethrin (74.8%) and lambda-cyhalothrin (87.5%) and Puerto Armuelles to permethrin (83%). Aedes aegypti El Empalme showed confirmed resistance to pirimiphos-methyl (62.3% mortality), chlorpyrifos-methyl (55.5%) and propoxur (85.3%). All strains of Ae. albopictus showed possible resistance to PYs and five strains to DDT. Only Ae. albopictus Canto del Llano showed confirmed resistance to pirimiphos-methyl (70% mortality) and malathion (62%). Esterase activity was variable across sites with the most frequent expression of α-EST compared to β-EST in Ae. aegypti populations. In Ae. Albopictus, the expressed enzymes were β-EST and MFOs. Through ANOVA, significant differences were established in the levels of enzymatic activity of α- and β-EST, MFOs and GST, with p < 0.001 in the Ae. aegypti and Ae. albopictus. The Kdr Val1016IIe mutation was detected in Ae. aegypti Aguadulce, El Coco and David. The odds ratio for the Val1016Ile mutation ranged from 0.8 to 20.8 in resistant mosquitoes, indicating the association between pyrethroid phenotypic resistance and the kdr mutation.ConclusionThe presence of a varied and generalized resistance, enzymatic mechanisms and the Val1016IIe mutation may be associated with the intensive use and possibly misuse of the different insecticides applied to control Aedes populations. These results highlight the need to develop a program for resistance management. Also, alternative approaches to mosquito control that do not involve insecticides should be explored.

Designing C9N10 Anchored Single Mo Atom as an Efficient Electrocatalyst for Nitrogen Fixation.

Electrochemical nitrogen reduction reaction (NRR) is a promising route for realizing green and sustainable ammonia synthesis under ambient conditions. However, one of the major challenges of currently available Single-atom catalysts (SACs) is poor catalytic activity and low catalytic selectivity, which is far away from the requirements of industrial applications. Herein, first-principle calculations within the density functional theory were performed to evaluate the feasibility of a single Mo atom anchored on a g-C9N10 monolayer (Mo@g-C9N10) as NRR electrocatalysts. The results demonstrated that the gas phase N2 molecule can be sufficiently activated on Mo@g-C9N10, and N2 reduction dominantly occurs on the active Mo atom via the preferred enzymatic mechanism, with a low limiting potential of -0.48 V. In addition, Mo@g-C9N10 possesses a good prohibition ability for the competitive hydrogen evolution reaction. More impressively, good electronic conductivity and high electron transport efficiency endow Mo SACs with excellent activity for electrocatalytic N2 reduction. This theoretical research not only accelerates the development of NRR electrocatalysts but also increases our insights into optimizing the catalytic performance of SACs.

New Trichoderma Strains Suppress Blue Mold in Oranges by Damaging the Cell Membrane of Penicillium italicum and Enhancing Both Enzymatic and Non-Enzymatic Defense Mechanisms in Orange Fruits

Blue mold disease, caused by Penicillium italicum (P. italicum), presents a significant challenge to orange fruits (Citrus sinensis L.) and other citrus crops globally. Biological control, particularly Trichoderma species, offers a promising alternative to synthetic fungicides. Therefore, this study aimed to isolate, identify, and evaluate the antagonistic activities of two Trichoderma isolates against P. italicum. These isolates were molecularly identified and assigned accession numbers PP002254 and PP002272, respectively. Both isolates demonstrated significant antifungal activity in dual culture assays. Moreover, the culture filtrates (CFs) of Trichoderma longibrachiatum PP002254 and Trichoderma harzianum PP002272 suppressed the mycelial growth of P. italicum by 77.22% and 71.66%, respectively. Additionally, CFs reduced the severity of blue mold on orange fruits by 26.85% and 53.81%, compared to 100% in the control group. Scanning electron microscopy revealed that treated P. italicum hyphae were shrunken and disfigured. Enzyme activities (catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase) in treated oranges increased, along with total soluble phenolics and flavonoids. Conversely, malondialdehyde (MDA) levels decreased in treated fruits. These findings suggest that T. longibrachiatum PP002254 and T. harzianum PP002272 could be effective biocontrol agents for managing blue mold and other citrus postharvest diseases.

Tutorial Review on the Set-Up and Running of Quantum Mechanical Cluster Models for Enzymatic Reaction Mechanisms.

Enzymes turnover substrates into products with amazing efficiency and selectivity and as such have great potential for use in biotechnology and pharmaceutical applications. However, details of their catalytic cycles and the origins surrounding the regio- and chemoselectivity of enzymatic reaction processes remain unknown, which makes the engineering of enzymes and their use in biotechnology challenging. Computational modelling can assist experimental work in the field and establish the factors that influence the reaction rates and the product distributions. A popular approach in modelling is the use of quantum mechanical cluster models of enzymes that take the first- and second coordination sphere of the enzyme active site into consideration. These QM cluster models are widely applied but often the results obtained are dependent on model choice and model selection. Herein, we show that QM cluster models can give highly accurate results that reproduce experimental product distributions and free energies of activation within several kcal mol-1, regarded that large cluster models with >300 atoms are used that include key hydrogen bonding interactions and charged residues. In this tutorial review, we give general guidelines on the set-up and applications of the QM cluster method and discuss its accuracy and reproducibility. Finally, several representative QM cluster model examples on metal-containing enzymes are presented, which highlight the strength of the approach.

Structural Insights and Catalytic Mechanism of 3-Hydroxybutyryl-CoA Dehydrogenase from Faecalibacterium Prausnitzii A2-165.

Atopic dermatitis (AD) is characterized by a T-helper cell type 2 (Th2) inflammatory response leading to skin damage with erythema and edema. Comparative fecal sample analysis has uncovered a strong correlation between AD and Faecalibacterium prausnitzii strain A2-165, specifically associated with butyrate production. Therefore, understanding the functional mechanisms of crucial enzymes in the butyrate pathway, such as 3-hydroxybutyryl-CoA dehydrogenase of A2-165 (A2HBD), is imperative. Here, we have successfully elucidated the three-dimensional structure of A2HBD in complex with acetoacetyl-CoA and NAD+ at a resolution of 2.2Å using the PAL-11C beamline (third generation). Additionally, X-ray data of A2HBD in complex with acetoacetyl-CoA at a resolution of 1.9 Å were collected at PAL-XFEL (fourth generation) utilizing Serial Femtosecond Crystallography (SFX). The monomeric structure of A2HBD consists of two domains, N-terminal and C-terminal, with cofactor binding occurring at the N-terminal domain, while the C-terminal domain facilitates dimerization. Our findings elucidate the binding mode of NAD+ to A2HBD. Upon acetoacetyl-CoA binding, the crystal structure revealed a significant conformational change in the Clamp-roof domain (root-mean-square deviation of 2.202 Å). Notably, residue R143 plays a critical role in capturing the adenine phosphate ring, underlining its significance in substrate recognition and catalytic activity. The binding mode of acetoacetyl-CoA was also clarified, indicating its lower stability compared to NAD+. Furthermore, the conformational change of hydrophobic residues near the catalytic cavity upon substrate binding resulted in cavity shrinkage from an open to closed conformation. This study confirms the conformational changes of catalytic triads involved in the catalytic reaction and presents a proposed mechanism for substrate reduction based on structural observations.

Physiology of malate dehydrogenase and how dysregulation leads to disease.

Malate dehydrogenase (MDH) is pivotal in mammalian tissue metabolism, participating in various pathways beyond its classical roles and highlighting its adaptability to cellular demands. This enzyme is involved in maintaining redox balance, lipid synthesis, and glutamine metabolism and supports rapidly proliferating cells' energetic and biosynthetic needs. The involvement of MDH in glutamine metabolism underlines its significance in cell physiology. In contrast, its contribution to lipid metabolism highlights its role in essential biosynthetic processes necessary for cell maintenance and proliferation. The enzyme's regulatory mechanisms, such as post-translational modifications, underscore its complexity and importance in metabolic regulation, positioning MDH as a potential target in metabolic dysregulation. Furthermore, the association of MDH with various pathologies, including cancer and neurological disorders, suggests its involvement in disease progression. The overexpression of MDH isoforms MDH1 and MDH2 in cancers like breast, prostate, and pancreatic ductal adenocarcinoma, alongside structural modifications, implies their critical role in the metabolic adaptation of tumor cells. Additionally, mutations in MDH2 linked to pheochromocytomas, paragangliomas, and other metabolic diseases emphasize MDH's role in metabolic homeostasis. This review spotlights MDH's potential as a biomarker and therapeutic target, advocating for further research into its multifunctional roles and regulatory mechanisms in health and disease.

Ubiquitin ligase signalling networks shape presynaptic development, function and disease.

Ubiquitin ligases are important regulators of nervous system development, function and disease. To date, numerous ubiquitin ligases have been discovered that regulate presynaptic biology. Here, we discuss recent findings on presynaptic ubiquitin ligases that include members from the three major ubiquitin ligase classes: RING, RBR and HECT. Several themes emerge based on findings across a range of model systems. A cadre of ubiquitin ligases is required presynaptically to orchestrate development and transmission at synapses. Multiple ubiquitin ligases deploy both enzymatic and non-enzymatic mechanisms, and act as hubs for signalling networks at the synapse. Both excitatory and inhibitory presynaptic terminals are influenced by ligase activity. Finally, there are several neurodevelopmental disorders and neurodegenerative diseases associated with presynaptic ubiquitin ligases. These findings highlight the growing prominence and biomedical relevance of the presynaptic ubiquitin ligase network.

Molecular insights into the structure and function of the Staphylococcus aureus fatty acid kinase

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. This complex consists of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, and is known to impact virulence and disease outcomes. Despite some recent studies, there remains many outstanding questions as to the enzymatic mechanism and structure of FAK . To better address this gap in knowledge, we used a combination of modeling, biochemical, and cell-based approaches to build on prior proposed models and identify critical details of FAK activity. Using bio-layer interferometry, we demonstrated nanomolar affinity between FakA and FakB that also indicates that FakA is dimer when binding FakB. Additionally, targeted mutagenesis of the FakA Middle domain demonstrates it possesses a metal binding pocket that is critical for FakA dimer stability and FAK function in vitro and in vivo. Lastly, we solved structures of the apo and ligand-bound FakA kinase domain to capture the molecular changes in the protein following ATP binding and hydrolysis. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

In Silico Analysis of the Molecular Interaction between Anthocyanase, Peroxidase and Polyphenol Oxidase with Anthocyanins Found in Cranberries.

Anthocyanins are bioactive compounds responsible for various physiological processes in plants and provide characteristic colors to fruits and flowers. Their biosynthetic pathway is well understood; however, the enzymatic degradation mechanism is less explored. Anthocyanase (β-glucosidase (BGL)), peroxidase (POD), and polyphenol oxidase (PPO) are enzymes involved in degrading anthocyanins in plants such as petunias, eggplants, and Sicilian oranges. The aim of this work was to investigate the physicochemical interactions between these enzymes and the identified anthocyanins (via UPLC-MS/MS) in cranberry (Vaccinium macrocarpon) through molecular docking to identify the residues likely involved in anthocyanin degradation. Three-dimensional models were constructed using the AlphaFold2 server based on consensus sequences specific to each enzyme. The models with the highest confidence scores (pLDDT) were selected, with BGL, POD, and PPO achieving scores of 87.6, 94.8, and 84.1, respectively. These models were then refined using molecular dynamics for 100 ns. Additionally, UPLC-MS/MS analysis identified various flavonoids in cranberries, including cyanidin, delphinidin, procyanidin B2 and B4, petunidin, pelargonidin, peonidin, and malvidin, providing important experimental data to support the study. Molecular docking simulations revealed the most stable interactions between anthocyanase and the anthocyanins cyanidin 3-arabinoside and cyanidin 3-glucoside, with a favorable ΔG of interaction between -9.3 and -9.2 kcal/mol. This study contributes to proposing a degradation mechanism and seeking inhibitors to prevent fruit discoloration.

Starch-related structural basis and enzymatic mechanism of the different appearances of soft rice

To investigate the fine starch structure characteristics and formation mechanism of high-quality appearance soft rice, two high-quality and low-quality soft rice varieties (HA-SR and LA-SR, respectively) were selected. Differences in appearance quality, fine starch structure, and activity of key enzymes involved in starch synthesis during the grain-filling stage were compared. The results showed that compared with LA-SR, HA-SR were less chalky, more transparent, had larger starch grains, a lower content of shorter chains (DP 6–24), a higher content of longer chains (DP ≥ 25), lower relative crystallinity, fewer ordered structures, more amorphous structures and larger thicknesses of semi-crystalline lamellae. In terms of amylase activity during the grain-filling stage, the AGPase and GBSS activities of HA-SR were higher, and the SBE activity of HA-SR was lower compared to LA-SR. In conclusion, higher AGPase activity can produce a higher filling rate resulting in fuller starch grain in soft rice. Fuller starch grains reduce the chalkiness of soft rice. Higher AGPase and GBSS activities and lower SBE activity can result in soft rice with more long-branched and less short-branched amylopectin. Thus, soft rice has lower relative crystallinity and less ordered structure. These structures may facilitate reduce grain chalkiness and improve grain transparency.

Understanding the Functional Dynamics of the TokK Enzyme in Carbapenem Biosynthesis via MD Simulations and QM/MM Calculations.

Due to the recent surge in antibiotic resistance, developing novel antibiotics is the demand of the time, and thus, a precise understanding of the catalytic mechanisms of enzymes involved in antibiotic biosynthesis becomes crucial. Here, we present a comprehensive investigation into the catalytic mechanism of TokK, a freshly characterized B12-dependent RSMT enzyme that plays an important role in carbapenem biosynthesis. Using MD simulations, we show how the plasticity of the active site facilitates substrate recognition while the quantum mechanics/molecular mechanics calculations provide a detailed mechanistic understanding of the methyl transfer process, elucidating stereochemical preferences. Notably, we demonstrate the indispensable role of Trp215 in orchestrating the proper orientation of the 5'-dA• radical for efficient substrate methylation, which strongly correlates with the previous findings where the mutation of Trp215 has severely affected the enzyme activity.

Effect of climate, crop, and management on soil phosphatase activity in croplands: A global investigation and relationships with crop yield

Agricultural and livestock production cover more than a third of the Earth's land surface and are crucial to food supply. Soil extracellular enzymes play an important role in the transformation of elements and compounds in soil, particularly acid (ACP) and alkaline (ALP) phosphatases (both, APases). These enzymes have a vital role in releasing phosphorus (P) from organic matter. However, the effect of climate variables and agro-ecosystem management on APase activity in croplands remains unclear, as does its eventual relationship with agricultural productivity. Therefore, we compiled a global database of APase activity in croplands (between 1977 and 2022) and we analysed 5876 observations across 474 papers to study climate variables, crop family, and management effects on ACP and ALP activity, and their relationship with yield. ACP activity is reduced by higher temperatures (p<0.001) and lower rainfall (p=0.002). There was an interaction effect of temperature and precipitation on ALP activity (p=0.046), with the negative effect of temperature being stronger with high precipitation, and low precipitation showing low ALP activity levels at any temperature. The crop family greatly influenced APase activity (p<0.001). Management practices affected ACP and ALP activity differently; ACP activity was positively influenced by organic fertilization combined with, crop rotation or irrigation by an average of 15.6 % and 30.7 %, respectively. ALP activity was mainly positively influenced by the interaction of two different factors: organic or inorganic-organic fertilization and reduced or zero tillage. Further understanding of soil enzyme mechanisms would aid global food security and yield. As ACP activity doubles from 100.0 to 200.0 mg pNP kg−1h−1, the crop yield increases by more than two-fold, an outcome not demonstrated in croplands until now. These results could enhance yield potential through the promotion of APase activity, and the consideration of climate variables and agro-ecosystem management, which could ultimately improve cost-benefit ratios for sustainable crop growth.