An official website of the United States government
The role of protein folding in prenucleation clusters on the activity of enzyme@metal–organic frameworks
Two steps must be satisfied to achieve high activity enzyme@MOFs: proper enzyme folding and low MOF crystallinity.
more »
« less
- Award ID(s):
- 2102033
- PAR ID:
- 10543036
- Publisher / Repository:
- Royal Society of Chemistry
- Date Published:
- Journal Name:
- Journal of Materials Chemistry A
- Volume:
- 12
- Issue:
- 2
- ISSN:
- 2050-7488
- Page Range / eLocation ID:
- 813 to 823
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Rieske dioxygenases are multi-component enzyme systems, naturally found in many soil bacteria, that have been widely applied in the production of fine chemicals, owing to the unique and valuable oxidative dearomatization reactions they catalyze. The range of practical applications for these enzymes in this context has historically been limited, however, due to their limited substrate scope and strict selectivity. In an attempt to overcome these limitations, our research group has employed the tools of enzyme engineering to expand the substrate scope or improve the reactivity of these enzyme systems in specific contexts. Traditionally, enzyme engineering campaigns targeting metalloenzymes have avoided mutations to metal-coordinating residues, based on the assumption that these residues are essential for enzyme activity. Inspired by the success of other recent enzyme engineering reports, our research group investigated the potential to alter or improve the reactivity of Rieske dioxygenases by altering or eliminating iron coordination in the active site of these enzymes. Herein, we report the modification of all three iron-coordinating residues in the active site of toluene dioxygenase both to alternate residues capable of coordinating iron, and to a residue that would eliminate iron coordination. The enzyme variants produced in this way were tested for their activity in the cis-dihydroxylation of a small library of potential aromatic substrates. The results of these studies demonstrated that all three iron-coordinating residues, in their natural state, are essential for enzyme activity in toluene dioxygenase, as the introduction of any mutations at these sites resulted in a complete loss of cis-dihydroxylation activity.more » « less
-
Farha, Omar (Ed.)Metal-Organic Frameworks (MOFs) are advanced platforms for enzyme immobilization. Enzymes can be entrapped via either diffusion (into pre-formed MOFs) or co-crystallization. Enzyme co-crystallization with specific metals/ligands in the aqueous phase, also known as biomineralization, minimizes the enzyme loss as compared to organic phase co-crystallization, removes the size limitation on enzymes and substrates, and can potentially broaden the application of enzyme@MOF composites. However, not all enzymes are stable/functional in the presence of excess metal ions and/or ligands currently available for co-crystallization. Furthermore, most current biomineralization-based MOFs have limited (acid-) pH stability, making it necessary to explore other metal-ligand combinations that can also immobilize enzymes. Here, we report our discovery on the combination of five metal ions and two ligands that can form biocomposites with two model enzymes differing in size and hydrophobicity in the aqueous phase under ambient conditions. Surprisingly, most of the formed composites are single- or multi- phase crystals even though the reaction phase is aqueous, with the rest as amorphous powders. All 20 enzyme@MOF composites showed good to excellent reusability, and were stable under weakly acidic pHs. The stability under weakly basic conditions depended on the selection of enzyme and metal-ligand combinations, yet for both enzymes, 3-4 MOFs offered decent stability under basic conditions. This work initiates the expansion of the current “library” of metal-ligand selection for encapsulating/biomineralizing large enzymes/enzyme clusters, leading to customized encapsulation of enzymes according to enzymes stability, functionality, and optimal pH.more » « less
-
Confining proteins in synthetic nanoscale spatial compartments has offered a cell-free avenue to understand enzyme structure–function relationships and complex cellular processes near the physiological conditions, an important branch of fundamental protein biophysics studies. Enzyme confinement has also provided advancement in biocatalysis by offering enhanced enzyme reusability, cost-efficiency, and substrate selectivity in certain cases for research and industrial applications. However, the primary research efforts in this area have been focused on the development of novel confinement materials and investigating protein adsorption/interaction with various surfaces, leaving a fundamental knowledge gap, namely, the lack of understanding of the confined enzymes (note that enzyme adsorption to or interactions with surfaces differs from enzyme confinement as the latter offers an enhanced extent of restriction to enzyme movement and/or conformational flexibility). In particular, there is limited understanding of enzymes' structure, dynamics, translocation (into biological pores), folding, and aggregation in extreme cases upon confinement, and how confinement properties such as the size, shape, and rigidity affect these details. The first barrier to bridge this gap is the difficulty in “penetrating” the “shielding” of the confinement walls experimentally; confinement could also lead to high heterogeneity and dynamics in the entrapped enzymes, challenging most protein-probing experimental techniques. The complexity is raised by the variety in the possible confinement environments that enzymes may encounter in nature or on lab benches, which can be categorized to rigid confinement with regular shapes, rigid restriction without regular shapes, and flexible/dynamic confinement which also introduces crowding effects. Thus, to bridge such a knowledge gap, it is critical to combine advanced materials and cutting-edge techniques to re-create the various confinement conditions and understand enzymes therein. We have spearheaded in this challenging area by creating various confinement conditions to restrict enzymes while exploring experimental techniques to understand enzyme behaviors upon confinement at the molecular/residue level. This review is to summarize our key findings on the molecular level details of enzymes confined in (i) rigid compartments with regular shapes based on pre-formed, mesoporous nanoparticles and Metal–Organic Frameworks/Covalent-Organic Frameworks (MOFs/COFs), (ii) rigid confinement with irregular crystal defects with shapes close to the outline of the confined enzymes via co-crystallization of enzymes with certain metal ions and ligands in the aqueous phase (biomineralization), and (iii) flexible, dynamic confinement created by protein-friendly polymeric materials and assemblies. Under each case, we will focus our discussion on (a) the way to load enzymes into the confined spaces, (b) the structural basis of the function and behavior of enzymes within each compartment environments, and (c) technical advances of our methodology to probe the needed structural information. The purposes are to depict the chemical physics details of enzymes at the challenging interface of natural molecules and synthetic compartment materials, guide the selection of enzyme confinement platforms for various applications, and generate excitement in the community on combining cutting-edge technologies and synthetic materials to better understand enzyme performance in biophysics, biocatalysis, and biomedical applications.more » « less
-
The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life. SHMT is a pyridoxal 5’-phosphate (PLP) - dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site. Here, we have characterized a Pro to Ser amino acid variant (P285S) that affects this conserved cis proline in soybean SHMT8. P285S was identified as one of a set of mutations that affect the resistance of soybean to the agricultural pathogen soybean cyst nematode. We find that replacement of Pro285 by serine eliminates PLP-mediated catalytic activity of SHMT8, reduces folate binding, decreases enzyme stability, and affects the dimer-tetramer ratio of the enzyme in solution. Crystal structures at 1.9 – 2.2 Å resolution reveal a local reordering of the polypeptide chain that extends an a-helix and shifts a turn region into the active site. This results in a dramatically perturbed PLP-binding pose, where the ring of the cofactor is flipped by ~180° with concomitant loss of conserved enzyme-PLP interactions. A nearby region of the polypeptide becomes disordered, evidenced by missing electron density for ~10 residues. These structural perturbations are consistent with the loss of enzyme activity and folate binding and underscore the important role of the Pro285 cis-peptide in SHMT structure and function.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D3TA05397K
