Multiple Enzymatic Reactions Research Articles

Zinc intake, SLC30A8 rs3802177 polymorphism, and colorectal cancer risk in a Korean population: a case-control study.

Zinc is an essential micronutrient involving in multiple enzymatic reactions of human metabolism and biological functions affecting the cancer development. However, the relationship between dietary zinc intake and colorectal cancer (CRC) risk has been unclear. Herein, our study investigated the relationship between dietary zinc intake and CRC risk, and examined how the SLC30A8 rs3802177 genetic variant affects this association. A total of 1431 CRC cases and 2704 controls were selected to investigate the relationship between dietary zinc intake and CRC risk. After excluding individuals without genotype data, 1097 CRC cases and 1559 controls were used to evaluate the interaction between dietary zinc intake and the rs3802177 polymorphism in CRC risk. The odds ratios (ORs) and 95% confidence intervals (CIs) were measured using unconditional logistic regression models. Higher dietary zinc intake was inversely associated with the risk of CRC in the total population [adjusted OR (aOR) = 0.80, 95% CI 0.66-0.96, p for trend = 0.018]. In the codominant model, G+ carriers of the SLC30A8 rs3802177 with higher consumption of zinc were observed to have a significantly lower risk of CRC in all participants (p for interaction = 0.020). In females, GG carriers with higher zinc intake showed a stronger protective effect against the development of CRC (p for interaction = 0.008). In summary, our findings suggest an inverse association between dietary zinc intake and CRC risk, and this relationship may be modified by SLC30A8 rs3802177 polymorphism.

The Role of Iron in Phytopathogenic Microbe-Plant Interactions: Insights into Virulence and Host Immune Response.

Iron is an essential element required for the growth and survival of nearly all forms of life. It serves as a catalytic component in multiple enzymatic reactions, such as photosynthesis, respiration, and DNA replication. However, the excessive accumulation of iron can result in cellular toxicity due to the production of reactive oxygen species (ROS) through the Fenton reaction. Therefore, to maintain iron homeostasis, organisms have developed a complex regulatory network at the molecular level. Besides catalyzing cellular redox reactions, iron also regulates virulence-associated functions in several microbial pathogens. Hosts and pathogens have evolved sophisticated strategies to compete against each other over iron resources. Although the role of iron in microbial pathogenesis in animals has been extensively studied, mechanistic insights into phytopathogenic microbe-plant associations remain poorly understood. Recent intensive research has provided intriguing insights into the role of iron in several plant-pathogen interactions. This review aims to describe the recent advances in understanding the role of iron in the lifestyle and virulence of phytopathogenic microbes, focusing on bacteria and host immune responses.

Phosphatidic acid: from biophysical properties to diverse functions.

Phosphatidic acid (PA), the simplest phospholipid, acts as a key metabolic intermediate and second messenger that impacts diverse cellular and physiological processes across species ranging from microbes to plants and mammals. The cellular levels of PA dynamically change in response to stimuli, and multiple enzymatic reactions can mediate its production and degradation. PA acts as a signalling molecule and regulates various cellular processes via its effects on membrane tethering, enzymatic activities of target proteins, and vesicular trafficking. Because of its unique physicochemical properties compared to other phospholipids, PA has emerged as a class of new lipid mediators influencing membrane structure, dynamics, and protein interactions. This review summarizes the biosynthesis, dynamics, and cellular functions and properties of PA.

Serum sodium as a diabetes risk factor?

Electrolytes play a key role in controlling fluid status, muscle contraction, neurological function, coagulation, and acid base status, as well as multiple enzymatic reactions. Diabetes is associated with multiple significant electrolyte disorders, predominantly affecting serum sodium (Na), potassium, and magnesium.1 In healthy individuals, serum Na levels range between 135–146 mmol/L. Hyponatremia is defined as a serum Na < 135 mmol/L, typically accompanied by a low serum osmolality (<275 mosm/L). It is the most frequently seen electrolyte disorder, present in about 5% of adults and 35% of hospitalized patients.2, 3 In a New York health system analysis of 10 385 patients hospitalized with COVID-19 in 2020, hyponatremia was present in 37.5% of patients.4 Low serum Na may activate the renin-angiotensin-aldosterone system, may cause increased sympathetic nervous system tone, and may be associated with oxidative stress. Insulin, either administered therapeutically or in the setting of hyperinsulinemia from insulin resistance, increases renal tubular water reabsorption via a presumed vasopressin effect and can result in hyponatremia. Chronic kidney disease and diuretic use in hypertension further increase the risk of hyponatremia as well as other electrolyte imbalances such as hypokalemia and hypomagnesemia.7 Serum Na simply may represent the state of hydration. Might some of these mechanisms contribute to effects of even small deviations in serum Na from normal? Should we look more carefully at Na as a biomarker of outcome? Further analysis of large clinical data sets may give answers to these questions.

Disruption of Intracellular Metal Homeostasis As a Vulnerability in SF3B1 Mutated AML

Acute Myeloid Leukemia (AML) is a heterogeneous disease in which outcome is highly dependent on cytogenetics and mutational profile. Adverse risk AML, recently defined and updated in the 2022 European LeukemiaNet (ELN) genetic risk classification, is associated with a poor overall survival. To identify novel therapeutic approaches for AML, we screened a library of 10,000 compounds across 56 genetically diverse primary AML specimens. We then looked for active compounds with increased activity towards adverse risk AML samples and with low cytotoxicity to normal hematopoietic cells. Among the hits identified, we were able to validate that S767 molecule is preferentially active on adverse risk AML samples. Additionally, we found a strong association between SF3B1 mutations and sensitivity to S767. To gain insights into S767 mechanism of action, we performed a CRISPR/Cas9 genome-wide functional screen, and identified multiples genes involved in mitochondrial processes both in synthetic rescue and synthetic lethal genes. In particular, knock-down of enzymes involved in heme biogenesis and iron-sulfur cluster biosynthesis appear to interfere with S767 cytotoxic activity. Given the central role of iron in these two synthetic pathways, and the structure of S767, we hypothesized that our molecule binds and disrupts intracellular iron homeostasis. Using UV-spectrometry analysis, we determined that S767 has metal affinity and binds to Fe2+, but also Cu2+ and Zn2+. We then quantified intracellular metal concentrations by ICP-MS (inductively coupled plasma mass spectrometry) and found that S767 exposure significantly decreases intracellular iron concentrations while increasing that of copper. Since metals are critical cellular cofactors involved in multiple enzymatic reactions, disruption of intracellular metal homeostasis can affect many processes. Mitochondrial respiratory chain is particularly sensitive to these perturbations as copper and iron-sulfur clusters are present in complexes I-IV of the electron transport chain and are essential for mitochondrial complexes function. By measuring oxygen consumption rate in leukemic cells exposed to S767, we found that the molecule indeed disrupts mitochondrial respiration. SF3B1 is a splicing factor recurrently mutated in myeloid malignancies including myelodysplastic syndromes (MDS) and AML. Heterozygous mutations typically affect SF3B1 splicing functions and induces consequently mis-splicing of target mRNAs, including the ABCB7 iron-sulfur cluster transporter. Analysis of the Leucegene transcriptomic data of primary AML specimens revealed mis-splicing and downregulation of ABCB7 in SF3B1 mutated patients. Strikingly, ABCB7 was one of the top synthetic lethal gene identified by chemogenomic CRISPR/Cas9 screen following exposure to S767 molecule. Given that ABCB7 exports iron-sulfur clusters out of the mitochondria to be assembled by cytosolic proteins and plays a role in iron homeostasis, we hypothesized that its aberrant expression in SF3B1 mutated AML participates to the high sensitivity of these leukemias to S767. We thus identified a novel molecule with selective activity in AML. Our data suggest that S767 is not a protein-targeting molecule but rather acts through the disruption of intracellular metal homeostasis, and ultimately affects mitochondrial respiration. Its mechanism of action highlighted a vulnerability in SF3B1 mutated AML which present a defect in ABCB7 expression. Importantly, SF3B1 mutations are now recognized in the 2022 ELN risk classification as an adverse prognostic marker if they are associated with intermediate or adverse risk AML, giving a unique opportunity to develop novel therapeutic approaches for this subgroup of patients with poor clinical outcome. In addition, SF3B1 mutations are found in 25% of MDS cases, which are associated with ring sideroblasts and accumulation of mitochondrial iron, and that could also benefit from such therapeutic approach.

A direct enzymatic evaluation platform (DEEP) to fine-tuning pyridoxal 5'-phosphate-dependent proteins for cadaverine production.

Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.

The complex role of kininogens in hereditary angioedema.

Human high molecular weight kininogen (HK) is the substrate from which bradykinin is released as a result of activation of the plasma “contact” system, a cascade that includes the intrinsic coagulation pathway, and a fibrinolytic pathway leading to the conversion of plasminogen to plasmin. Its distinction from low molecular weight kininogen (LK) was first made clear in studies of bovine plasma. While early studies did suggest two kininogens in human plasma also, their distinction became clear when plasma deficient in HK or both HK and LK were discovered. The light chain of HK is distinct and has the site of interaction with negatively charged surfaces (domain 5) plus a 6th domain that binds either prekallikrein or factor XI. HK is a cofactor for multiple enzymatic reactions that relate to the light chain binding properties. It augments the rate of conversion of prekallikrein to kallikrein and is essential for the activation of factor XI. It indirectly augments the “feedback” activation of factor XII by plasma kallikrein. Thus, HK deficiency has abnormalities of intrinsic coagulation and fibrinolysis akin to that of factor XII deficiency in addition to the inability to produce bradykinin by factor XII-dependent reactions. The contact cascade binds to vascular endothelial cells and HK is a critical binding factor with binding sites within domains 3 and 5. Prekallikrein (or factor XI) is attached to HK and is brought to the surface. The endothelial cell also secretes proteins that interact with the HK-prekallikrein complex resulting in kallikrein formation. These have been identified to be heat shock protein 90 (HSP 90) and prolylcarboxypeptidase. Cell release of urokinase plasminogen activator stimulates fibrinolysis. There are now 6 types of HAE with normal C1 inhibitors. One of them has a mutated kininogen but the mechanism for overproduction (presumed) of bradykinin has not yet been determined. A second has a mutation involving sulfation of proteoglycans which may lead to augmented bradykinin formation employing the cell surface reactions noted above.

Retraction: Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions

Retraction: Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions

Retraction: Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions.

Retraction: Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions.

The Redox Active [2Fe-2S] Clusters: Key-Components of a Plethora of Enzymatic Reactions—Part I: Archaea

The earliest forms of life (i.e., Archaea, Bacteria, and Eukarya) appeared on our planet about ten billion years after its formation. Although Archaea do not seem to possess the multiprotein machinery constituted by the NIF (Nitrogen Fixation), ISC (Iron Sulfur Cluster), SUF (sulfur mobilization) enzymes, typical of Bacteria and Eukarya, some of them are able to encode Fe-S proteins. Here we discussed the multiple enzymatic reactions triggered by the up-to-date structurally characterized members of the archaeal family that require the crucial presence of structurally characterized [2Fe-2S] assemblies, focusing on their biological functions and, when available, on their electrochemical behavior.

MicroRNA miR-8 promotes cell growth of corpus allatum and juvenile hormone biosynthesis independent of insulin/IGF signaling in Drosophila melanogaster

The Drosophila melanogaster corpus allatum (CA) produces and releases three types of sesquiterpenoid hormones, including juvenile hormone III bisepoxide (JHB3), juvenile hormone III (JH III), and methyl farnesoate (MF). JH biosynthesis involves multiple discrete enzymatic reactions and is subjected to a comprehensive regulatory network including microRNAs (miRNAs). Using a high throughput sequencing approach, we have identified abundant miRNAs in the D. melanogaster ring gland, which consists of the CA, prothoracic gland, and corpus cardiaca. Genetic and qPCR screens were then performed in an attempt to uncover the full repertoire of CA miRNAs that are involved in regulating metamorphosis. miR-8 was identified as a potential candidate and further studied for its role in the CA. Overexpression of miR-8 in the CA increased cell size of the gland and expression of Jhamt (a gene coding for a key regulatory enzyme in JH biosynthesis), resulting in pupal lethality. By contrast, sponge-mediated reduction of miR-8 in the CA decreased cell size and Jhamt expression, but did not cause lethality. Further investigation revealed that miR-8 promotes cell growth independent of insulin/IGF signaling. Taken together, these experiments show that miR-8 is highly expressed in the CA and exerts its positive effects on cell growth and JH biosynthesis. The miRNAs data in the ring gland also provide a useful resource to study how miRNAs collaboratively regulate hormone synthesis in D. melanogaster.

Phosphatidic acid: an emerging versatile class of cellular mediators.

Lipids function not only as the major structural components of cell membranes, but also as molecular messengers that transduce signals to trigger downstream signaling events in the cell. Phosphatidic acid (PA), the simplest and a minor class of glycerophospholipids, is a key intermediate for the synthesis of membrane and storage lipids, and also plays important roles in mediating diverse cellular and physiological processes in eukaryotes ranging from microbes to mammals and higher plants. PA comprises different molecular species that can act differently, and is found in virtually all organisms, tissues, and organellar membranes, with variations in total content and molecular species composition. The cellular levels of PA are highly dynamic in response to stimuli and multiple enzymatic reactions can mediate its production and degradation. Moreover, its unique physicochemical properties compared with other glycerophospholipids allow PA to influence membrane structure and dynamics, and interact with various proteins. PA has emerged as a class of new lipid mediators modulating various signaling and cellular processes via its versatile effects, such as membrane tethering, conformational changes, and enzymatic activities of target proteins, and vesicular trafficking.

Elaboration of the Corticosteroid Synthesis Pathway in Primates through a Multistep Enzyme.

Metabolic networks are complex cellular systems dependent on the interactions among, and regulation of, the enzymes in the network. Although there is great diversity of types of enzymes that make up metabolic networks, the models meant to understand the possible evolutionary outcomes following duplication neglect specifics about the enzyme, pathway context, and cellular constraints. To illuminate the mechanisms that shape the evolution of biochemical pathways, I functionally characterize the consequences of gene duplication of an enzyme family that performs multiple subsequent enzymatic reactions (a multistep enzyme) in the corticosteroid pathway in primates. The products of the corticosteroid pathway (aldosterone and cortisol) are steroid hormones that regulate metabolism and stress response in tetrapods. These steroid hormones are synthesized by a multistep enzyme Cytochrome P450 11B (CYP11B) that performs subsequent steps on different carbon atoms of the steroid derivatives. Through ancestral state reconstruction and in vitro characterization, I find that the primate ancestor of the CYP11B1 and CYP11B2 paralogs had moderate ability to synthesize both cortisol and aldosterone. Following duplication in Old World primates, the CYP11B1 homolog specialized on the production of cortisol, whereas its paralog, CYP11B2, maintained its ability to perform multiple subsequent steps as in the ancestral pathway. Unlike CYP11B1, CYP11B2 could not specialize on the production of aldosterone because it is constrained to perform earlier steps in the corticosteroid synthesis pathway to achieve the final product aldosterone. These results suggest that enzyme function, pathway context, along with tissue-specific regulation, both play a role in shaping potential outcomes of metabolic network elaboration.

THE “OLD SAILORS “ ILLNESS MAKES A RETURN

Vitamin C, also known as ascorbic acid, is a co-factor in multiple enzymatic reactions including that of collagen synthesis. Due to the absence of the enzyme L-gulonolactone oxidase, humans are unable to synthesise ascorbic acid; hence it is recognised as an essential nutrient. Scurvy refers to the clinical presentation of a deficiency of ascorbic acid, which occurs as a result of inadequate dietary intake. It has long been considered an illness of historical rather than contemporary significance. However, a tendency towards Western diets rich in processed foods and lacking in fresh produce has given rise to the re-emergence of the condition in the developed world. Current evidence suggests that there is a resurgence of scurvy in Sydney. A case is highlighted of an otherwise healthy 35year old male who presented to a hospital emergency dental clinic with generalised red, boggy gingivae. Clinical examination revealed bilateral involvement of the buccal and lingual/palatal gingivae in both the mandibular and maxillary arches, predominantly affecting the interdental papillae. Radiographic examination confirmed there was no associated bone loss or bony pathology. The clinical differential diagnoses included leukaemia, Kaposi sarcoma or possible scurvy. A battery of serological investigations yielded the eventual diagnosis of severe ascorbic acid deficiency (vitamin C level

Dual phenazine gene clusters enable diversification during biosynthesis.

Biosynthetic gene clusters (BGCs) bridging genotype and phenotype continuously evolve through gene mutations and recombinations to generate chemical diversity. Phenazine BGCs are widespread in bacteria, and the biosynthetic mechanisms of the formation of the phenazine structural core have been illuminated in the last decade. However, little is known about the complex phenazine core-modification machinery. Here, we report the diversity-oriented modifications of the phenazine core through two distinct BGCs in the entomopathogenic bacterium Xenorhabdus szentirmaii, which lives in symbiosis with nematodes. A previously unidentified aldehyde intermediate, which can be modified by multiple enzymatic and non-enzymatic reactions, is a common intermediate bridging the pathways encoded by these BGCs. Evaluation of the antibiotic activity of the resulting phenazine derivatives suggests a highly effective strategy to convert Gram-positive specific phenazines into broad-spectrum antibiotics, which might help the bacteria-nematode complex to maintain its special environmental niche.

182 - Time-course metabolomic analysis of manganese toxicity reveals biomarkers of oxidative stress and amino acid metabolism as early cellular targets

Manganese (Mn) is a naturally occurring essential nutrient at low doses that functions as a cofactor in multiple enzymatic reactions, provides structural stability and participates in redox activities. However, high Mn exposure even under federal safety limits for air Mn levels, may cause a dose-dependent progression of Parkinsonism. The molecular mechanisms and critical transitions from normal Mn physiology to low dose toxicity as well as early targets of Mn toxicity nonetheless remains poorly understood. In this temporal study, we used human neuroblastoma SH-SY5Y cells exposed to Mn concentrations varied over a physiologic (5μM MnCl2) and toxicological range (50μM MnCl2) for 0, 0.5, 2, 5 and 10h (n=9 per Mn concentration and time point). Metabolomics analysis was done using liquid chromatography with ultra-high resolution mass spectrometry and cellular Mn accumulation was measured using ICP-MS. Results showed cellular Mn accumulation increased upto 5h in a dose dependent manner, beyond which Mn uptake was stabilized. Pathway enrichment analysis showed that methionine and cysteine metabolism; urea cycle metabolism; glycine, serine, alanine and threonine metabolism; and histidine metabolism were significantly altered within 30min of manganese treatment and persisted upto 10h suggesting these pathways define early targeted mechanisms of Mn toxicity. Discriminatory metabolites involved in methionine and cysteine pathway include methionine, homocysteine, 1,2 dihydroxy-3-keto-5-methylthiopentene and 5’-methylthioadenosine. Discriminatory metabolites involved in glycine, serine, alanine and threonine metabolism include 3-phosphohydroxypyruvate, phosphoserine, serine and creatine. These early responders may reflect rapid transport and redistribution of Mn within the cell. In conclusion, we demonstrate key metabolic biomarkers and pathways that respond dynamically to varying Mn concentration and attempt to provide a chronological fingerprint of Mn dependent toxicity. Time course metabolomics thereby provides a useful tool to predict metabolic status for dynamic systems at the cellular level in response to environmental chemical exposures.

Molecular Tunnels in Enzymes and Thermophily: A Case Study on the Relationship to Growth Temperature.

Developments in protein expression, analysis and computational capabilities are decisively contributing to a better understanding of the structure of proteins and their relationship to function. Proteins are known to be adapted to the growth rate of microorganisms and some microorganisms (named (hyper)thermophiles) thrive optimally at high temperatures, even above 100 °C. Nevertheless, some biomolecules show great instability at high temperatures and some of them are universal and required substrates and cofactors in multiple enzymatic reactions for all (both mesophiles and thermophiles) living cells. Only a few possibilities have been pointed out to explain the mechanisms that thermophiles use to successfully thrive under high temperatures. As one of these alternatives, the role of molecular tunnels or channels in enzymes has been suggested but remains to be elucidated. This study presents an analysis of channels in proteins (i.e., substrate tunnels), comparing two different protein types, glutamate dehydrogenase and glutamine phosphoribosylpyrophosphate amidotransferase, which are supposed to present a different strategy on the requirement for substrate tunnels with low and high needs for tunneling, respectively. The search and comparison of molecular tunnels in these proteins from microorganisms thriving optimally from 15 °C to 100 °C suggested that those tunnels in (hyper)thermophiles are required and optimized to specific dimensions at high temperatures for the enzyme glutamine phosphoribosylpyrophosphate amidotransferase. For the enzyme glutamate dehydrogenase, a reduction of empty spaces within the protein could explain the optimization at increasing temperatures. This analysis provides further evidence on molecular channeling as a feasible mechanism in hyperthermophiles with multiple relevant consequences contributing to better understand how they live under those extreme conditions.

A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula.

Bacterial cytochrome P450 monooxygenases (P450s) are promising biocatalysts for chemical syntheses because they catalyze a variety of oxidations on non-activated hydrocarbons using O2 . However, the requirement of two auxiliary proteins, an electron transfer protein and a reductase, for the catalysis is a major bottleneck for in vitro applications of these monooxygenases. The authors previous study showed that artificial assembly of a bacterial P450 with its auxiliary proteins using a heterotrimeric proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus yields a self-sufficient P450, but partial dissociation of P450 from the complex at catalytic concentrations reduces the apparent specific activity of this self-sufficient P450. In this study, a Metallosphaera sedula PCNA is used, which is currently the most stable heterotrimeric PCNA, to assemble a bacterial P450 with its auxiliary proteins at submicromolar protein concentrations. The apparent specific monooxygenase activity of the M. sedula PCNA-assembled P450 with auxiliary proteins is saturated at protein concentrations of 40 nM, and is 2.1-fold higher than that of the S. solfataricus PCNA-assembled P450. Therefore, M. sedula PCNA represents a versatile tool to facilitate multiple enzymatic reactions, including the P450 monooxygenase system.

Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions

AbstractNext‐generation therapeutic approaches are expected to rely on the engineering of biomimetic cellular systems that can mimic specific cellular functions. Herein, we demonstrate a highly effective route for constructing structural and functional eukaryotic cell mimics by loading pH‐sensitive polymersomes as membrane‐associated and free‐floating organelle mimics inside the multifunctional cell membrane. Metabolism mimicry has been validated by performing successive enzymatic cascade reactions spatially separated at specific sites of cell mimics in the presence and absence of extracellular organelle mimics. These enzymatic reactions take place in a highly controllable, reproducible, efficient, and successive manner. Our biomimetic approach to material design for establishing functional principles brings considerable enrichment to the fields of biomedicine, biocatalysis, biotechnology, and systems biology.

Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions.

Next-generation therapeutic approaches are expected to rely on the engineering of biomimetic cellular systems that can mimic specific cellular functions. Herein, we demonstrate a highly effective route for constructing structural and functional eukaryotic cell mimics by loading pH-sensitive polymersomes as membrane-associated and free-floating organelle mimics inside the multifunctional cell membrane. Metabolism mimicry has been validated by performing successive enzymatic cascade reactions spatially separated at specific sites of cell mimics in the presence and absence of extracellular organelle mimics. These enzymatic reactions take place in a highly controllable, reproducible, efficient, and successive manner. Our biomimetic approach to material design for establishing functional principles brings considerable enrichment to the fields of biomedicine, biocatalysis, biotechnology, and systems biology.