Computational insights into the binding modes, keto-enol tautomerization and stereo-electronically controlled decarboxylation of oxaloacetate in the active site of macrophomate synthase.

Oxaloacetic acid (OAA) is a ß-ketocarboxylic acid, which plays an important role as an intermediate in some metabolic pathways, including the tricarboxylic acid cycle, gluconeogenesis and fatty acid biosynthesis. Animal studies have indicated that supplementing oxaloacetic acid shows an increase of lifespan and other substantial health benefits including mitochondrial DNA protection, and protection of retinal, neural and pancreatic tissues. Most of the chemical transformations of OAA in the metabolic pathways have been extensively studied; however, the understanding of decarboxylation of OAA at the atomic level is relatively lacking. Here, we carried out MD simulations and combined quantum mechanical/molecular mechanical (QM/MM) calculations as an example to systematically elucidate the binding modes, keto-enol tautomerization and decarboxylation of OAA in the active site of macrophomate synthase (MPS), which is a Mg(II)-dependent bifunctional enzyme that catalyzes both the decarboxylation of OAA and [4+2] cycloaddition of 2-pyrone with the decarboxylated intermediate of OAA (pyruvate enolate). On the basis of our calculations, it was found that the Mg2+-coordinated oxaloacetate may exist in enol forms and keto forms. The four keto forms can be transformed into each other by simply rotating the C2-C3 single bond, nevertheless, the keto-enol tautomerization strictly requires the assistance of pocket water molecules. In addition, the decarboxylation is stereo-electronically controlled, i.e., it is the relative orientation of the terminal carboxyl anion that determines the rate of decarboxylation. As such, the chemistry of oxaloacetate in the active site of MPS is complex. On one hand, the most stable binding mode (K-I) may undergo enol-keto tautomerization to isomerize to the enol form, which may further react with the second substrate; on the other hand, K-I may isomerize to another binding mode K-II to proceed decarboxylation to generate pyruvate enolate and CO2. Starting from K-I, the enol-keto tautomerization corresponds to a barrier of 16.2 kcal mol-1, whereas the decarboxylation is associated with an overall barrier of 19.7 kcal mol-1. These findings may provide useful information for understanding the chemistry of OAA and the catalysis of related enzymes, and they are basically in agreement with the available experimental kinetic data.

Reverse N-Substituted Hydroxamic Acid Derivatives of Fosmidomycin Target a Previously Unknown Subpocket of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase (DXR).

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

Identifying a key spot for electron mediator-interaction to tailor CO dehydrogenase's affinity.

Fe‒S cluster-harboring enzymes, such as carbon monoxide dehydrogenases (CODH), employ sophisticated artificial electron mediators like viologens to serve as potent biocatalysts capable of cleaning-up industrial off-gases at stunning reaction rates. Unraveling the interplay between these enzymes and their associated mediators is essential for improving the efficiency of CODHs. Here we show the electron mediator-interaction site on ChCODHs (Ch, Carboxydothermus hydrogenoformans) using a systematic approach that leverages the viologen-reactive characteristics of superficial aromatic residues. By enhancing mediator-interaction (R57G/N59L) near the D-cluster, the strategically tailored variants exhibit a ten-fold increase in ethyl viologen affinity relative to the wild-type without sacrificing the turn-over rate (kcat). Viologen-complexed structures reveal the pivotal positions of surface phenylalanine residues, serving as external conduits for the D-cluster to/from viologen. One variant (R57G/N59L/A559W) can treat a broad spectrum of waste gases (from steel-process and plastic-gasification) containing O2. Decoding mediator interactions will facilitate the development of industrially high-efficient biocatalysts encompassing gas-utilizing enzymes.

Dynamic Metabolons Using Stimuli-Responsive Protein Cages.

Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical reactions in living cells to meet changing cellular needs. However, replicating such capability in synthetic metabolons remains a challenge due to our limited understanding of the mechanisms by which the assembly and disassembly of such naturally occurring multienzyme complexes are controlled. Here, we report the synthesis of chemical- and light-responsive protein cages for assembling synthetic metabolons, enabling the dynamic regulation of enzymatic reactions in living cells. Particularly, a chemically responsive domain was fused to a self-assembled protein cage subunit, generating engineered protein cages capable of displaying proteins containing cognate interaction domains on their surfaces in response to small molecular cues. Chemical-induced colocalization of sequential enzymes on protein cages enhances the specificity of the branched deoxyviolacein biosynthetic reactions by 2.6-fold. Further, by replacing the chemical-inducible domain with a light-inducible dimerization domain, we created an optogenetic protein cage capable of reversibly recruiting and releasing targeted proteins onto and from the exterior of the protein cages in tens of seconds by on-off of blue light. Tethering the optogenetic protein cages to membranes enables the formation of light-switchable, membrane-bound metabolons, which can repeatably recruit-release enzymes, leading to the manipulation of substrate utilization across membranes on demand. Our work demonstrates a powerful and versatile strategy for constructing dynamic metabolons in engineered living cells for efficient and controllable biocatalysis.

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Capturing the Binuclear Copper State of Peptidylglycine Monooxygenase Using a Peptidyl-Homocysteine Lure.

Peptidylglycine monooxygenase is a copper-dependent enzyme that catalyzes C-alpha hydroxylation of glycine extended pro-peptides, a critical post-translational step in peptide hormone processing. The canonical mechanism posits that dioxygen binds at the mononuclear M-center to generate a Cu(II)-superoxo species capable of H atom abstraction from the peptidyl substrate, followed by long-range electron tunneling from the CuH center. Recent crystallographic and biochemical data have challenged this mechanism, suggesting instead that an "open-to-closed" transition brings the copper centers closer, allowing reactivity within a binuclear intermediate. Here we present the first direct observation of an enzyme-bound binuclear copper species, captured by the use of an Ala-Ala-Phe-hCys inhibitor complex. This molecule reacts with the fully reduced enzyme to form a thiolate-bridged binuclear species characterized by EXAFS of the WT and its M314H variant and with the oxidized enzyme to form a novel mixed valence entity characterized by UV/vis and EPR. Mechanistic implications are discussed.

Affinity-assisted covalent self-assembly of PduQ-SpyTag and Nox-SpyCatcher to construct multi-enzyme complexes on the surface of magnetic microsphere modified with chelated Ni2.

It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni2+, was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (NC) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni2+ and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and NC at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni2+ chelating surface was proposed. The time evolution of NADH showed that the immobilized P-T/N-C complex formed by this method and the free P-T/N-C complex exhibited similar synergistic catalytic properties, and presented higher catalytic efficiency than the simple mixing of P-T and NC. The optimal catalytic conditions, stability and reusability of the immobilized multi-enzyme complexes prepared in this study were also discussed by comparing them with free enzymes. In this study, we demonstrate a simple and effective strategy for self-assembling SpyTag/SpyCatcher fusion proteins on the surface of magnetic beads, which is inspiring for the construction of more cascade enzyme systems at the interface. It provides a new method for facilitating the rapid construction of immobilized multi-enzyme complexes in vitro from the crude cell lysis.

Structural and functional properties of uridine 5'-monophosphate synthase from Coffea arabica.

In higher eukaryotes and plants, the last two sequential steps in the de novo biosynthesis of uridine 5'-monophosphate (UMP) are catalyzed by a bifunctional natural chimeric protein called UMP synthase (UMPS). In higher plants, UMPS consists of two naturally fused enzymes: orotate phosphoribosyltransferase (OPRTase) at N-terminal and orotidine-5'-monophosphate decarboxylase (ODCase) at C-terminal. In this work, we obtained the full functional recombinant protein UMPS from Coffea arabica (CaUMPS) and studied its structure-function relationships. A biochemical and structural characterization of a plant UMPS with its two functional domains is described together with the presentation of the first crystal structure of a plant ODCase at 1.4 Å resolution. The kinetic parameters measured of CaOPRTase and CaODCase domains were comparable to those reported. The crystallographic structure revealed that CaODCase is a dimer that conserves the typical fold observed in other ODCases from prokaryote and eukaryote with a 1-deoxy-ribofuranose-5'-phosphate molecule bound in the active site of one subunit induced a closed conformation. Our results add to the knowledge of one of the key enzymes of the de novo biosynthesis of pyrimidines in plant metabolism and open the door to future applications.

How Acyl Carrier Proteins (ACPs) Direct Fatty Acid and Polyketide Biosynthesis.

Megasynthases, such as type I fatty acid and polyketide synthases (FASs and PKSs), are multienzyme complexes responsible for producing primary metabolites and complex natural products. Fatty acids (FAs) and polyketides (PKs) are built by assembling and modifying small acyl moieties in a stepwise manner. A central aspect of FA and PK biosynthesis involves the shuttling of substrates between the domains of the multienzyme complex. This essential process is mediated by small acyl carrier proteins (ACPs). The ACPs must navigate to the different catalytic domains within the multienzyme complex in a particular order to guarantee the fidelity of the biosynthesis pathway. However, the precise mechanisms underlying ACP-mediated substrate shuttling, particularly the factors contributing to the programming of the ACP movement, still need to be fully understood. This Review illustrates the current understanding of substrate shuttling, including concepts of conformational and specificity control, and proposes a confined ACP movement within type I megasynthases.

Nano- and Microscale Confinements in DNA-Scaffolded Enzyme Cascade Reactions.

Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion-reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano- and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot-based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.

Synthetic Multienzyme Assemblies for Natural Product Biosynthesis.

In nature, enzymes that catalyze sequential reactions are often assembled as clusters or complexes. The formation of multienzyme complexes, or metabolons, brings the enzyme active sites into proximity to promote intermediate transfer, decrease intermediate leakage, and streamline the metabolic flux towards the desired products. We and others have developed synthetic versions of metabolons through various strategies to enhance the catalytic rates for synthesizing valuable chemicals inside microbes. Synthetic multienzyme complexes range from static enzyme nanostructures to dynamic enzyme coacervates. Enzyme complexation optimizes the metabolic fluxes inside microbes, increases the product titer, and supplies the field with high-yield microbe strains that are amenable to large-scale fermentation. Enzyme complexes constructed inside microbial cells can be separated as independent entities and catalyze biosynthetic reactions ex vivo; such a feature gains these complexes another name, "synthetic organelles" - new subcellular entities with independent structures and functions. Still, the field is seeking new strategies to better balance dynamicity and confinement and to achieve finer control of local compartmentalization in the cells, as the natural multienzyme complexes do. Industrial applications of synthetic multienzyme complexes for the large-scale production of valuable chemicals are yet to be realized. This review focuses on synthetic multienzyme complexes that are constructed and function inside microbial cells.

Carrier-Free Immobilization of Multi-Enzyme Complex Facilitates In†Vitro Synthetic Enzymatic Biosystem for Biomanufacturing.

A method is developed for carrier-free immobilization of multi-enzyme complexes with more than four enzymes by utilization of polypeptide interactions (SpyCatcher-SpyTag and dockerin-cohesin) and enzyme component self-oligomerization. Two pairs of scaffoldins with different arrangements of SpyCatcher-SpyTag and cohesins are prepared to recruit the four dockerin-containing cascade enzymes (i. e., alpha-glucan phosphorylase, phosphoglucomutase, inositol 1-phosphate synthase, and inositol 1-phosphatase) that can convert starch into inositol, forming multi-enzyme complexes. These self-assembled enzyme complexes show higher initial reaction rates than the four-enzyme cocktail. Moreover, water-insoluble self-assembled multi-enzyme complexes are observed, being the carrier-free immobilized multi-enzyme complex aggregates. These immobilized enzyme complexes can be recycled easily by simple centrifuging followed by resuspension for another round of reaction. Not only can these immobilized enzyme complexes be obtained by mixing the purified enzyme components, but also by the mixing of crude cell extracts. Therefore, the strategy for the carrier-free immobilization of enzyme complex sheds light on improving the catalytic capability of in vitro synthetic enzymatic biosystems.

Effects of phenolic compounds on 3ß-hydroxysteroid dehydrogenase activity in human and rat placenta: Screening, mode of action, and docking analysis.

Human 3ß-hydroxysteroid dehydrogenase type I (HSD3B1) and rat type IV (HSD3B4) in placentas catalyze the conversion of pregnenolone to progesterone, which plays a key role in maintaining pregnancy. Many phenolic compounds potentially inhibit HSD3B in placentas as endocrine disruptors. In this study, the effects of 16 phenolic compounds on the activity of human HSD3B1 and rat HSD3B4 were determined and the structure-activity relationship was compared. HSD3B1 in human placental microsomes and HSD3B4 in rat placental microsomes were used to measure their activities and pregnenolone and NAD+ were used as substrates. Of the 16 phenolic compounds, 4-nonylphenol, pentabromophenol, and 2-bromophenol resulted in residual human HSD3B1 activity lower than 50 % and 4-nonylphenol and pentabromophenol resulted in residual rat HSD3B4 activity lower than 50 %. 4-Nonylphenol, pentabromophenol, and 2-bromophenol were mixed inhibitors of human HSD3B1, with Ki values of 2.31, 3.58 and 4.86 µM, respectively, while 4-nonylphenol and pentabromophenol were noncompetitive inhibitors of rat HSD3B4 with Ki values of 20.86 and 141.8 µM. Molecular docking showed that 4-nonylphenol, pentabromophenol, and 2-bromophenol docked to the active sites of human HSD3B1 and rat HSD3B4, and the shift of residue S125 in human HSD3B1 to T125 in rat HSD3B4 could explain the species-dependent difference in their inhibitory potency and mode of action. This study demonstrates that 4-nonylphenol, pentabromophenol, and 2-bromophenol are mixed inhibitors of human placental HSD3B1, while 4-nonylphenol and pentabromophenol are noncompetitive inhibitors of rat HSD3B4, possibly blocking the placental steroidogenesis.

Modular structure of complex II: An evolutionary perspective.

Succinate dehydrogenases (SDHs) and fumarate reductases (FRDs) catalyse the interconversion of succinate and fumarate, a reaction highly conserved in all domains of life. The current classification of SDH/FRDs is based on the structure of the membrane anchor subunits and their cofactors. It is, however, unknown whether this classification would hold in the context of evolution. In this work, a large-scale comparative genomic analysis of complex II addresses the questions of its taxonomic distribution and phylogeny. Our findings report that for types C, D, and F, structural classification and phylogeny go hand in hand, while for types A, B and E the situation is more complex, highlighting the possibility for their classification into subgroups. Based on these findings, we proposed a revised version of the evolutionary scenario for these enzymes in which a primordial soluble module, corresponding to the cytoplasmatic subunits, would give rise to the current diversity via several independent membrane anchor attachment events.

Cryo-EM structure of the SEA complex.

The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.

Photoswitchable carbon-dot liposomes mediate catalytic cascade reactions for amplified dynamic treatment of tumor cells.

Most biochemical reactions that occur in living organisms are catalyzed by a series of enzymes and proceed in a tightly controlled manner. The development of artificial enzyme cascades that resemble multienzyme complexes in nature is of current interest due to their potential in various applications. In this study, a nanozyme based on photoswitchable carbon-dot liposomes (CDsomes) was developed for use in programmable catalytic cascade reactions. These CDsomes prepared from triolein are amphiphilic and self-assemble into liposome-like structures in an aqueous environment. CDsomes feature excitation-dependent photoluminescence and, notably, can undergo reversible switching between a fluorescent on-state and nonfluorescent off-state under different wavelengths of light irradiation. This switching ability enables the CDsomes to exert photocatalytic oxidase- and peroxidase-like activities in their on- (bright) and off- (dark) states, respectively, resulting in the conversion of oxygen molecules into hydrogen peroxide (H2O2), followed by the generation of active hydroxyl radicals (OH). The two steps of oxygen activation can be precisely controlled in a sequential manner by photoirradiation at different wavelengths. Catalytic reversibility also enables the CDsomes to produce sufficient reactive oxygen species (ROS) to effectively kill tumor cells. Our results reveal that CDsomes is a promising photo-cycling nanozyme for precise tumor phototherapy through regulated programmable cascade reactions.

Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

Thioester synthesis by a designed nickel enzyme models prebiotic energy conversion.

The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the NiI species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.

Self-Assembled Multienzyme Nanostructures for Biocatalysis in Cellulo.

Multienzyme complexes naturally exist in cells to catalyze cascade reactions in metabolic pathways. By clustering the enzymes in close proximity, these nanomachineries achieve effective conversion of metabolites. Bioengineers are working on the development of synthetic versions of multienzyme complexes in cells to synergize heterologous biosynthesis. Assembling enzymes on protein scaffolds through protein-protein interactions is a viable and facile way to form synthetic multienzyme complexes. Here, we describe the general methods to construct self-assembled multienzyme nanostructures in Escherichia coli for biosynthesis of valuable chemicals.

Fast and efficient DNA replication with purified human proteins.

Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase1-7. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.