Functional Multi-enzyme Complexes In Vitro
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Published: Thu, 24 May 2018
Molecular self-assembly offers a method of sophisticated materials constructed with precision. Designing self-assembling enzyme structures is of particular interest for the unique functional capabilities of enzymes, as shown in Figure 2. Chemically induced assembly has been shown to be a powerful tool for the investigation of cellular events and for its easy operation and low cost compared to bioconjuction. Chemical inducers can be cofactors, inhibitors, metal ions, which are based on specific interaction of molecule and enzyme. Chemical inducers bring the two enzymes together to form multi-enzyme. A number of reviews have covered the self-assembly of proteins and enzymes by chemicals. King N.P. et al discussed the principles employed in recent efforts to design complex and geometrically specific protein assemblies, with a focus on practical approaches. However, precise manipulation of protein self-assembly behavior in vitro is still a great challenge. Here we review recent studies in the chemical induced self-assembly of multi-enzyme system from the perspective of multi-enzyme complex organization, enzyme interactions, and regulation of assembly.
Inhibitor induced multi-enzyme assembly
Inhibitor induced dimerization has been reported as the controlled dimerization of proteins via dimerizers. During the process of dimerization, the dimerizers assemble proteins into homospecific or heterospecific multivalent nanostructures. An enzyme inhibitor binds with enzymes specifically and decreases their activity. Drug discovery typically focus on the identification and design of inhibitors to perturb enzyme function, which greatly depend on the chemical structure.
Carlson and co-workers reported self-assembly of wild-type Escherichia coli dihydrofolate reductase (DHFR) into protein nanorings using dimeric methotrexate molecules, which tethered together by a flexible peptide linker. The enzymes are capable of spontaneously forming highly stable cyclic structures with diameters ranging from 8 to 20 nm. The nanoring size is dependent on the length and composition of the peptide linker, on the affinity and conformational state of the dimerizer, and on induced protein-protein interactions.
Chou reported the preparation of dihydrofolate reductase (DHFR)-histidine triad nucleotide nanorings by chemically induced self-assembly. DHFR molecules with fused peptide chain of variable length were spontaneously self-assemble into protein macrocycles after treatment with a dimeric enzyme inhibitor, Bis-MTX-C9. The ring size, ranging in size from 10 to 70 nm, was dependent on the length and composition of the peptide linking the fusion proteins. The enzymatic efficiencies for the monomer and intramolecular macrocycle were found to be nearly identical, while the larger dimeric nanoring was found to have a modestly lower kcat/Km value. The nanorings catalytic efficiency was dependent on ring size, which indicated that the arrangement of supermolecular assemblies of enzymes may be used to control their catalytic parameters. However, the activator used for multi-enzyme assembly has not been reported before, which can greatly improve enzyme activity and may have greatly potential in multi-enzyme biosynthesis.
Cofactor induced multi-enzyme assembly
Cofactor-dependent enzymes, such as oxidoreductases and transferases, intramolecularly assembly of enzyme subunits by cofactor binding have been widely reported. Cofactor as a small molecular for enzyme catalysis.
Cofactors can also be used for inducing multi-enzyme assembly. Bis-NAD+ has been reported for affinity precipitation of dehydrogenases in 1980s. Mansson et al used bis-NAD+ analogue to locate lactate dehydrogenase and alcohol dehydrogenase face to face and then cross-linked of the two enzymes with glutaraldehyde on agarose beads. The study of site-to-site directed immobilization effect improve the NADH production from 19% to 50%, which indicated that the NADH was preferentially channeled to lactate dehydrogenase due to the positioned active sites of the two enzymes.
Similar work reported by Siegbahn as the bi-enzyme complex was formed by crosslinking lactate dehydrogenase and alcohol dehydrogenase with glutaraldehyde, which indicated an enhancement of 1.36 fold of the NADH regeneration when lactate dehydrogenase and alcohol dehydrogenase were site-to-site oriented.
Cofactor induced assembly can form the site-to-site oriented structure, has the advantage easy operation and maintains the enzymes’ activity maintain. However, the interaction of NAD+ with enzyme is relatively low.
Metal ions induced multi-enzyme assembly
Metal ions guide proteins into forming large assemblies, which provide a wide platform to modulate the metal coordination environment through distant, noncovalent interactions, exactly as natural metalloproteins and enzymes do. Metal ions in metalloenzymes located in the pocket whose shape fits the substrate, which are usually coordinated by nitrogen, oxygen or sulfur centers belonging to amino acid residues. Since approximately half of all proteins contain a metal ion, metal ions induced enzyme assembly is a promising method. Metal ions induced protein assembly is recently hot topic. There are two main types of metal ions induced protein assembly, namely, metal ions chelating sites on the artificial His-tags of enzymes and chelating sites on the surface of enzymes.
His-tagging is the most widespread strategy to purify recombinant proteins. With the addition of 4-10 poly-histidine tag to the N terminus or C terminus of a target protein, the tagged protein purification was achieved by immobilized metal affinity chromatography. Multi-enzyme complex were formed with the Ni2+ and bis-His coordination of GDH-NOX fused enzymes, which enhanced enzyme activity and stability for the biosynthesis of DHA from glycerol with cofactor regeneration..
Chelating sites on the surface of enzymes
The metal ions coordinated with the chelating sites on the surface of proteins was reported. Chelating sites should be on the surfaces to coordinate with metal ions, and the interfaces where chelating sites are located should be complementary to form stable self-assemblies. Yushi Bai, et al [Bai, Y.S. et al. Highly ordered protein nanorings designed by accurate control of glutathione S-transferase self-assembly. J Am Chem Soc 135, 10966-10969 (2013). ]reported a variant of glutathione S-transferase (sjGST-2His) which has two properly oriented His metal-chelating sites on the surface self-assembled in a fixed bending manner to form protein nanorings. The accurate orientation of proteins and self-assembly was based on metal-ion-chelating interactions and nonspecific protein–protein interactions. This work provides a de novo design strategy for the construction of novel protein superstructures. The self-assembly of glutathione S-transferase into nanowires was also reported[Zhang, W. et al. Self-assembly of glutathione S-transferase into nanowires. Nanoscale 4, 5847-5851 (2012).].
Designed metal coordination interactions to arrange enzyme into highly ordered supramolecular architectures has been reported recently[Salgado, E.N., Radford, R.J. & Tezcan, F.A. Metal-Directed Protein Self-Assembly. Accounts Chem Res 43, 661-672 (2010).].Enzymes represent particularly attractive building blocks due to their chemical and structural versatility, for new and improved supramolecular properties. Metal-directed enzyme self-assembly yields stable architectures and high catalysis efficiency. These emergent physical and functional properties are attained with minimal modification of the original building blocks
Brodin reported the self-assembly of a designed variant of cytochrome cb(562) by zinc ion coordination to uniform 1D nanotubes or 2D arrays with very high chemical stabilities. Their metal-mediated frameworks was used as the templated growth of small Pt-0 nanocrystals. [Brodin, J.D., Carr, J.R., Sontz, P.A. & Tezcan, F.A. Exceptionally stable, redox-active supramolecular protein assemblies with emergent properties. P Natl Acad Sci USA 111, 2897-2902 (2014).]
Bogdan et al reported [Bogdan, N.D. et al. Metal Ion Mediated Self-Assembly Directed Formation of Protein Arrays. Biomacromolecules 12, 3400-3405 (2011).] the self-assembled inorganic–protein arrays by FeII complexation of protein-conjugated terpyridine units (ligand) to form well-defined and controllable size and structure. Residue-specific conjugation between the complexing unit (terpy) containing an activity-based probe and a corresponding active enzyme (papain) performed on this unique building block (ligand) leads to chemical species of unprecedented constitution.
Metal ion induced assembly are controllable by environmental factors that affect the coordination or reactivity of the metal ion: the presence of the metal itself, external chelators, pH, and the solution redox state. Thus, metal ions can augment or provide all three essential properties of proteins as nature’s favorite build-ing blocks: structure, chemical reactivity, and stimuli- responsiveness.
Metal ions are frequently found in natural protein-protein interfaces, where they stabilize quaternary or supramolecular protein structures, mediate transient protein-protein interactions, and serve as catalytic centers. Paralleling these natural roles, coordination chemistry of metal ions is being increasingly utilized in creative ways toward engineering and controlling the assembly of functional supramolecular peptide and protein architectures. Here we provide a brief overview of this emerging branch of metalloprotein/peptide engineering and highlight a few select examples from the recent literature that best capture the diversity and future potential of approaches that are being developed.
Conclusions and Outlook
Constructing functional multi-enzyme complexes in vitro by mimicking the natural enzyme complex has great biotechnological potentials in metabolic engineering, multi-enzyme-mediated biocatalysis, and cell-free synthetic pathway biotransformation. This review summarizes chemically assembling of multi-enzymes based on the affinity included by small molecular, namely, cofactor, substrate, inhibitor, and metal ions, et al. Distinctions were made based on the assembling driving force, structure of multi-enzyme complexes and mechanism of catalytic efficiency enhancement. Furthermore, the current challenges of multi-enzyme assembly in vitro induced by chemicals was addressed and gave an outlook on future developments.
In this review, a classification of multi-enzyme assembly methods is proposed. Special emphasis is placed on the description of constructing functional multi-enzyme complexes by small molecular induced self-assembly. Assembling of multi-enzymes based on the affinity induced by small molecular, namely, cofactor, inhibitor, and metal ions were discussed. Furthermore, the advantage and disadvantage of each method from the reaction and process considerations are described.
A variety of approaches for multi-enzymatic synthesis in vivo using biological systems or in vitro with isolated biocatalysts have been successfully used for the synthesis of complex molecules, especially the chiral chemicals which frequently are not readily accessible by chemical synthesis. In the long term, multi-enzyme processes will replace many chemically catalyzed processes. Biocatalysis today is growing not only in the fine chemicals and pharmaceuticals but also in the production of bulk chemicals. The relevant multi-enzyme catalysis processes have a significant potential for industrial application.
Several challenges remain for multi-enzyme processes despite the strong drivers for greener and ever more effective chemical process technology.Multi-enzyme assembly into exquisite, complex, yet highly ordered architectures is challenging due to the complexity of enzyme structures and interactions. Consequently, the prediction of multi-enzyme complex configurations, the structure controlled assembly and the dynamic kinetic simulation of assembly process are also challenging. Current efforts aim at the prediction of multi-enzyme complex configurations as well as at nanoscale reconstruction, and control of cascade reaction. The design of multi-enzymatic systems based on the structure controlling and function prediction. In Nature’s hierarchy such design and engineering studies can provide useful information. New approaches that allow the controlled assembly of multiple enzymes at a nanometer scale with precisely structure and function will increase reaction rates and the efficiency of longer synthetic enzymatic cascades. Another frontier in multi-enzyme synthesis is the design of multi-step processes, involving mathematical modeling, process technology, and protein engineering. By viewing multi-enzyme assembly process in terms of structure and function relationship, it is possible to unify a diverse range of investigations, highlights their interrelationships, and see routes.
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