Multienzyme Complex Research Articles

CRISPR/Cas-directed programmable assembly of multi-enzyme complexes.

We describe a versatile CRISPR/Cas-based strategy to construct multi-enzyme complexes scaffolded on a DNA template in programmable patterns. Catalytically inactive dCas9 nuclease was used in combination with SpyCatcher-SpyTag chemistry to assemble enzymes in a highly modular fashion. Five enzymes comprising the violacein biosynthesis pathway were precisely organized in nanometer proximity; a notable increase in violacein production demonstrated the benefits of scaffolding.

Cohesin-Dockerin Code in Cellulosomal Dual Binding Modes and Its Allosteric Regulation by Proline Isomerization

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule Fluorescence Resonance Energy Transfer and molecular dynamics simulations on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

In cellulo FRET-FLIM and single molecule tracking reveal the supra-molecular organization of the pyoverdine bio-synthetic enzymes in Pseudomonas aeruginosa.

The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.

OGDHL silencing promotes hepatocellular carcinoma by reprogramming glutamine metabolism

Mitochondrial dysfunction and subsequent metabolic deregulation are commonly observed in cancers, including hepatocellular carcinoma (HCC). When mitochondrial function is impaired, reductive glutamine metabolism is a major cellular carbon source for de novo lipogenesis to support cancer cell growth. The underlying regulators of reductively metabolized glutamine in mitochondrial dysfunction are not completely understood in tumorigenesis. We systematically investigated the role of oxoglutarate dehydrogenase-like (OGDHL), one of the rate-limiting components of the key mitochondrial multi-enzyme OGDH complex (OGDHC), in the regulation of lipid metabolism in hepatoma cells and mouse xenograft models. Lower expression of OGDHL was associated with advanced tumor stage, significantly worse survival and more frequent tumor recurrence in 3 independent cohorts totaling 681postoperative HCC patients. Promoter hypermethylation and DNA copy deletion of OGDHL were independently correlated withreduced OGDHL expression in HCC specimens. Additionally, OGDHL overexpression significantly inhibited the growth of hepatoma cells in mouse xenografts, while knockdown of OGDHL promoted proliferation of hepatoma cells. Mechanistically, OGDHL downregulation upregulated the α-ketoglutarate (αKG):citrate ratio by reducing OGDHC activity, which subsequently drove reductive carboxylation of glutamine-derived αKG via retrograde tricarboxylic acid cycling in hepatoma cells. Notably, silencing of OGDHL activated the mTORC1 signaling pathway in anαKG-dependent manner, inducing transcription of enzymes with key roles in de novo lipogenesis. Meanwhile, metabolic reprogramming in OGDHL-negative hepatoma cells provided an abundant supply of NADPH and glutathione to support the cellular antioxidant system. The reduction of reductive glutamine metabolism through OGDHL overexpression or glutaminase inhibitors sensitized tumor cells to sorafenib, a molecular-targeted therapy for HCC. Our findings established that silencing of OGDHL contributed to HCC development and survival by regulating glutamine metabolic pathways. OGDHL is a promising prognostic biomarker and therapeutic target for HCC. Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and is correlated with a high mortality rate. In patients with HCC, lower expression of the enzyme OGDHL is significantly associated with worse survival. Herein, we show that silencing of OGDHL induces lipogenesis and influences the chemosensitization effect of sorafenib in liver cancer cells by reprogramming glutamine metabolism. OGDHL is a promising prognostic biomarker and potential therapeutic target in OGDHL-negative liver cancer.

Nwd1 Regulates Neuronal Differentiation and Migration Through Purinosome Formation in the Developing Cerebral Cortex

Engagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat d omain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction.

Tryptophan synthase (TS) is a heterotetrameric αββα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αββα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.

Effects of Direct Fed-Microorganisms and Enzyme Blend Co-Administration on Growth Performance in Broilers Fed Diets With or Without Antibiotics

SUMMARY Direct-Fed Microorganisms (DFM) and exogenous enzymes have been demonstrated to improve growth performance in poultry and are potentially important components of management programs for antibiotic-free (ABF) poultry production. In this study, we evaluated the effect of a feed additive composed of a DFM product containing spores of 3 Bacillus amyloliquefaciens strains and a multi-enzyme complex of xylanase, amylase, and protease on the performance of broilers with or without an antibiotic growth promoter (AGP), either bacitracin methylene disalicylate (BMD) or virginiamycin. Growth performance of broilers fed one of 6 treatments arranged in a 3 (AGP) × 2 (additive) factorial was evaluated over a 42 d production period. Overall, administration of the feed additive improved body weight, body weight gain, and feed conversion ratio. Administration of either antibiotic significantly reduced cumulative FCR when compared to the control, while BMD administration reduced foot pad lesions as compared to the control. Our results suggest the co-administration of DFM and enzymes may be an important component of ABF poultry production programs and the benefits of their use are not dependent on the presence or absence of AGP.

Plant Polysaccharide Xyloglucan and Enzymes That Hydrolyze It (Review)

Various types of plant raw material are widely used in the pulp and paper, textile, food, and agricultural industries and pharmacology. One of the problems of utilizing the complex plant biomass is poor knowledge of its hemicellulose content and the lack of effective enzymes for hydrolysis of hemicelluloses. Xyloglucan is the major structural and storage polysaccharide in all dicots and a great number of monocots. It has a branched architecture with a backbone constructed of β-1,4-connected cellotetraose units decorated with short sidechains composed of xylose, galactose, arabinose, fucose and some other residues. Sidechain composition and alternation order are specie-specific and can change during cell growth resulting in variety of xyloglucan structural types. In general, xyloglucan structure depends on the taxonomic position of the plant. Structural features of xyloglucans belonging to different taxonomic groups are discussed in the evolutionary aspect. Xyloglucan hydrolysis is a necessary condition during conversion of plant biomass into high added-value products. Variety of xyloglucan structural types complicates the selection of enzymes for its hydrolysis. Xyloglucanase-containing multienzyme complexes can be used for efficient decomposition of plant biomass polysaccharides into fermentable sugars for biotechnology, and for the improvement of feed quality. Investigation of xyloglucanases is necessary for the development of methods for protecting plants from pathogenic microorganisms, which use these enzymes in the invasion of plant tissue. The article discusses structural features of xyloglucans from different taxonomic groups in the evolutionary aspect and reviews selection of xyloglucanases for efficient hydrolysis of complex plant biomass.

Isolation and subunit structure of the xylanosome complex produced by Actinotalea fermentans JCM9966.

Xylanosomes, also known as hemicellulosomes, are hemicellulose-degrading nano-scale multienzyme complexes produced by some Firmicutes, Actinobacteria, and Fungi. Here we report the isolation of the MECs produced by Actinotalea fermentas JCM9966, as well as the functional studies and subunit structure revealed by proteomic identifications. The isolated MECs here shows similar particle size with the xylanosomes produced by C. cellulans F16, have several conserved multi-domain proteins, while differ significantly in enzymatic activities and low molecular weight subunit compositions, indicating diverse capability as well as bias in degrading hemicelluloses.

The properties of the linker in a mini-scaffoldin influence the catalytic efficiency of scaffoldin-mediated enzyme complexes

Synthetic enzyme complexes have been successfully used to accelerate the reaction rate of cascade enzyme biocatalysis. Protein scaffold-mediated enzyme complexes are often constructed by assembling cascade enzymes on the artificial protein scaffoldin to form sophisticated biomimetic architectures and enhance the catalytic efficiency of biocatalytic processes. However, the effects of the linker in scaffoldin on the performance of the enzyme complexes have not been clarified. In this study, a scaffoldin-mediated two-enzyme complex containing triosephosphate isomerase (TIM) and fructose-1,6-bisphosphate aldolase/phosphatase (FBPA) was constructed, and the initial production rate of fructose 6-phosphate (F6P) was determined with different types of fine-tuning linkers. Enzyme complexes with linker length of 25 amino acids in scaffoldin exhibited the highest initial F6P production rate compared with linker length of 0, 10, or 57 amino acids in scaffoldin. This result indicated that an appropriate interdomain spacing between functional domains was required by multienzyme complexes to facilitate effective cascade catalysis. Then, the most popular flexible linker GGGGS (unit F) and rigid linker EAAAK (unit R) were introduced into this 25 amino acid linker to investigate the effect of linker flexibility on the initial reaction rate of the TIM–FBPA enzyme complex. The synthetic enzyme complex with the semirigid linker FRRRF in scaffoldin showed the highest initial F6P production rate of 10.16 μM/min, which indicates that the linker's amino acid composition in scaffoldin may lead to significant changes in the spatial architecture of the TIM–FBPA complex and consequently affect the initial reaction rate. Precise linker length and flexibility allow an appropriate interdomain conformation to enable efficient cascade reactions. Collectively, our results showed that fine-tuning the initial reaction rate of enzyme complexes is an integrated systematic engineering, including adjusting the multienzyme architecture, linker length, and linker flexibility, which provides rational guidance for designing effective multienzyme complexes in the future.

Selection of the Optimal Enzyme Composition for Sugar Beet Pulp Conversion

It was shown that the presence of cellobiohydrolase, β-glucosidase, endoglucanase, arabinoxylan-arabinofuranhydrolase, pectin lyase, and polygalacturonase is necessary for efficient hydrolysis of sugar beet pulp (SBP). Optimal multienzyme complexes consisting of the same enzymes and additional endoarabinase or exoarabinase, endogalactanase, β-xylosidase, endoxylanase, and/or α-arabinofuranosidase were determined. These components enabled the conversion of SBP based on a yield of total reducing sugars (RSs) of 61–68%, an arabinose yield of 94%, and a glucose yield of 63–79%. The optimal complex from dry multienzyme preparations (EP) of cellulases, hemicellulases, and pectinases, which are produced by the fungal strains Penicillium canescens, P. verruculosum, and Aspergillus foetidus, enabled SBP hydrolysis based on arabinose and glucose yields close to 100% at an initial SBP concentration of 100–250 g/L and an EP concentration of 5–10 mg protein/g SBP after 24–48 h of hydrolysis.

SpyCatcher/SpyTag-Mediated Self-Assembly of a Supramolecular Complex for Improved Biocatalytic Production of Trehalose

Trehalose has been utilized in the food, cosmetic, and pharmaceutical industries due to its ability to stabilize biomolecules. In this study, we developed a strategy of constructing a self-assembling supramolecular multienzyme complex for the production of trehalose using SpyCatcher and SpyTag. Maltooligosyl trehalose synthase (MTS) and maltooligosyl trehalose trehalohydrolase (MTH) were fused to SpyCatcher and its corresponding ligand, SpyTag. The fused MTS-SpyCatcher (MTS-SC) and MTH-SpyTag (MTH-ST) self-assembled into a supramolecular multienzyme complex (MTS-MTH) via the isopeptide bonds formed between SpyCatcher and SpyTag. MTS-MTH showed the faster reaction rate, which reached the peak of trehalose conversion rate 10 h earlier than the free enzymes. The results demonstrate that the multienzyme self-assembly via the protein-peptide interactions of SpyCatcher and SpyTag can increase the catalytic efficiency of sequential reactions.

Molecular characterization of hypothetical scaffolding-like protein S1 in multienzyme complex produced by Paenibacillus curdlanolyticus B-6

Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a hypothetical scaffolding-like protein and several xylanases and cellulases. The largest (280-kDa) component protein, called S1, has cellulose-binding ability and xylanase activity, thus was considered to function like the scaffolding proteins found in cellulosomes. S1 consists of 863 amino acid residues with predicted molecular mass 91,029 Da and includes two N-terminal surface layer homology (SLH) domains, but most of its sequence shows no homology with proteins of known function. Native S1 (nS1) was highly glycosylated. Purified nS1 and recombinant Xyn11A (rXyn11A) as a major xylanase subunit could assemble in a complex, but recombinant S1 (rS1) could not interact with rXyn11A, indicating that S1 glycosylation is necessary for assembly of the multienzyme complex. nS1 and rS1 showed weak, typical endo-xylanase activity, even though they have no homology with known glycosyl hydrolase family enzymes. S1 and its SLH domains bound tightly to the peptide-glycan layer of P. curdlanolyticus B-6, microcrystalline cellulose, and insoluble xylan, indicating that the SLHs of S1 bind to carbohydrate polymers and the cell surface. When nS1 and rXyn11A were co-incubated with birchwood xylan, the degradation ability was synergistically increased compared with that for each protein; however synergy was not observed for rS1 and rXynA. These results indicate that S1 may have a scaffolding protein-like function by interaction with enzyme subunits and polysaccharides through its glycosylated sites and SLH domains.

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action.

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.

Isolation and subunit compositions of the xylanosome complexes produced by Cellulosimicrobium species

Cellulosimicrobium cellulans, which is type species of the genus Cellulosimicrobium, produces xylanase predominant nanoscale multienzyme complexes, i.e., xylanosomes, when grown on water-insoluble polysaccharides. Here, we report on the isolation of similar multienzyme complexes (MECs) produced by two other species in genus Cellulosimicrobium (Cellulosimicrobium funkei and Cellulosimicrobium terreum). Functional studies and subunit structure identifications using genomic sequencing and proteomic techniques were also performed. When compared with the xylanosomes produced by C. cellulans F16, the isolated MECs showed a larger particle size and shared at least three conserved multidomain proteins. In addition, they also exhibited different enzymatic activities and subunit compositions, which indicates diverse capability and strategies in degrading hemicelluloses.

Perspectives on the use of lipoic acid in the support of disease treatment*

Lipoic acid (LA) is a natural compound present in food and used as a dietary supplement. LA is endogenously synthetized in small amounts from octanoid acid in the mitochondria. This compound occurs naturally in vegetables such as spinach, broccoli and in animal tissues, in the kidneys, heart and liver. It has been shown that LA is a cofactor in the multienzyme complexes that are responsible for oxidative decarboxylation of α- ketoacids. LA and its reduced form, dihydrolipoic acid (DHLA), have many biological functions leading to a wide variety of actions such as anti-inflammation and antioxidant protection, scavenging of reactive oxygen species, regenerating other antioxidant agents, such as vitamins C and E, and cytosolic glutathione, chelating the transitional metal ions (e.g. iron and copper), and modulating the signal transduction of nuclear factor. Many authors regard LA as a potentially useful agent in the treatment and/or prevention of many diseases such as diabetes mellitus, overweight, obesity, hypertension, heart diseases, inflammation. This review concentrates on the role of LA in the treatment of diabetes mellitus, obesity, inflammation and blood pressure regulation. LA can be considered as a potentially useful drug in treatment of many diseases, particularly those related to excessive production of free radicals.

Peculiarities of the metabolism in broiler chickens under the introduction of multienzyme complex “Rovabio” in the diet

The article presents data on studying effects of the enzyme preparation “Rovabio” in rye-containing diets for broilers of the cross “Smena-7” at the age of 14 to 42 days. It was revealed that the inclusion of the enzyme preparation “Rovabio” in the dose of 50 g/t in the combination with trace elements CoCO3 (0.57 mg/kg of feed), Cr2(SO4)3*6H2O (0.38 mg/kg of feed) with the replacement of 15% of wheat to 15% of rye into the diet demonstrated a positive impact of the used dosages of the trace elements on digestibility of organic matter (6.49%), crude protein (4.59%), the protein synthesis and stimulated hematopoiesis in birds. The multienzyme complex increases the integrity of chickens to 96.7% and profitability of poultry meat production by 2.0% due to endo-1.4-ß-xylanase and endo-1.3(4)-ß-glucanase.

Overexpression of a Novel Cytochrome P450 Promotes Flavonoid Biosynthesis and Osmotic Stress Tolerance in Transgenic Arabidopsis.

Flavonoids are mainly associated with growth, development, and responses to diverse abiotic stresses in plants. A growing amount of data have demonstrated the biosynthesis of flavonoids through multienzyme complexes of which the membrane-bounded cytochrome P450 supergene family shares a crucial part. However, the explicit regulation mechanism of Cytochrome P450s related to flavonoid biosynthesis largely remains elusive. In the present study, we reported the identification of a stress-tolerant flavonoid biosynthetic CtCYP82G24 gene from Carthamus tinctorius. The transient transformation of CtCYP82G24 determined the subcellular localization to the cytosol. Heterologously expressed CtCYP82G24 was effective to catalyze the substrate-specific conversion, promoting the de novo biosynthesis of flavonoids in vitro. Furthermore, a qRT-PCR assay and the accumulation of metabolites demonstrated that the expression of CtCYP82G24 was effectively induced by Polyethylene glycol stress in transgenic Arabidopsis. In addition, the overexpression of CtCYP82G24 could also trigger expression levels of several other flavonoid biosynthetic genes in transgenic plants. Taken together, our findings suggest that CtCYP82G24 overexpression plays a decisive regulatory role in PEG-induced osmotic stress tolerance and alleviates flavonoid accumulation in transgenic Arabidopsis.

Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD–RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

Self-Assembled Multienzyme Nanostructures on Synthetic Protein Scaffolds.

Sequential enzymes in a biosynthetic pathway often self-assemble to form nanomachineries known as multienzyme complexes inside cells. Enzyme self-assembly insulates toxic intermediates, increases the efficiency of intermediate transfer, minimizes metabolic crosstalk, streamlines flux, and improves the product yield. Artful structures and superior catalytic functions of these natural nanomachines inspired the development of synthetic multienzyme complexes to expedite biosynthesis. Here we present a versatile self-assembly strategy to construct multienzyme nanostructures based on synthetic protein scaffolds. The protein scaffolds were formed using the spontaneous protein reaction of SpyCatcher and SpyTag. Two types of protein scaffolds were generated: two skeleton proteins cross-linked and hierarchically assembled into heterogeneous nanostructures (the cross-linked scaffold), and head-to-tail cyclization of a dual-reactive skeleton protein gave a homogeneous cyclic scaffold. Sequential enzymes from the menaquinone biosynthetic pathway were assembled on both scaffolds through the docking domain interactions derived from polyketide synthases. Both scaffolded assemblies effectively increased the yield of the final product of the cascade catalytic reaction in menaquinone biosynthesis. Surprisingly, the rate enhancements were driven by different mechanisms: the cross-linked scaffold assembly streamlined the overall flow of the reactants, whereas the cyclic scaffold assembly accelerated the catalytic efficiency of the rate-limiting enzyme. Altogether, self-assembly of sequential enzymes by combining the SpyCatcher/SpyTag reaction and the docking domain interactions yielded protein-based nanostructures with special architecture, exceptional catalytic activity, and unexpected catalytic mechanisms. This work demonstrates a versatile strategy of gaining more powerful biocatalysts by protein self-assembly for efficient bioconversion of valuable chemicals.