Tuning properties of biocatalysis using protein cage architectures

Abstract

Compartmentalization of cellular activities is an extremely important mechanism within cells, across all domains of life, for high efficiency of cell function. Bacterial microcompartments are exemplary protein-based cage structures that...

Tuning Multistep Biocatalysis through Enzyme and Cofactor Colocalization in Charged Porous Protein Macromolecular Frameworks

“…We want to note that, although the mobility of NAD-(+36)GFP plays an important role in the multistep reaction in the PMF, the reaction did proceed at very low ionic strengths, such as I = 37 mM (albeit with inhibited multistep efficiency compared to free enzymes, as shown in Figure S6). It is likely that some population of the partitioned NAD-(+36)GFP was located in proximity to both ER and PtDH within the enzyme–PMF materials, such that the NAD moiety could shuttle between the neighboring enzyme pair via a “swing arm”-like mechanism , without the translocation of the (+36)GFP moiety. However, increased mobility could endow a larger population of NAD-(+36)GFP with access to more enzymes within the materials (instead of merely neighboring enzymes), increasing the overall usage of NAD-(+36)GFP as the multistep intermediate.…”

“…Both this result and the activity measurements on individual enzymes (Figure S7) suggest that the partitioning process of the enzymes into the PMF, which provides a highly charged environment (the enzymes were immobilized on the highly charged P22 VLP exterior), did not significantly change enzyme activities. Furthermore, due to the high porosity (i.e., large interstitial space), the PMF does not pose any diffusion barrier to small molecules, such as unmodified NAD, , whose diffusion is much faster than the turnover rates of most enzymes ,, including PtDH and ER . Consequently, these control experiments with unmodified NAD also suggest that, when it can diffuse freely into and out of the PMF, the cofactor was equally active to both the partitioned and the free enzymes.…”

“…Because of the high cost of the cofactors for acellular catalysis, recent work has focused on multienzyme systems that replenish the cofactor stock, where a cofactor-regenerating enzyme is coupled with a cofactor-consuming enzyme capable of catalyzing a reaction of interest. , However, these systems still require investment of a large quantity of cofactor to maintain a high overall rate . Tethering cofactors (especially NAD) to different supports (such as nanoparticles or the enzymes themselves) has been a popular approach in the biocatalysis field, as it allows recycling and reuse of cofactor molecules more efficiently. − To further enhance the utilization–regeneration process of cofactor at low quantity, substrate channeling has been proposed in the design of biocatalytic materials. , Compared to conventional multienzyme cascades (no channeling), inducing channeling of a cofactor requires the directed transfer of the cofactor between an enzyme couple without its diffusion into the bulk solution, which can potentially increase the effective local concentration of the cofactor available to the enzymes. As a result, even with a very small input quantity of cofactor, the system can still operate at high catalytic rates.…”

“…Previous theoretical analyses ,, and experimental verification − suggest that, due to fast diffusion of small molecules in aqueous solution, interenzyme proximity in biocatalytic complexes does not automatically lead to substrate channeling. Instead, substrate channeling is usually achieved by actively controlling the diffusion of the multistep intermediates, by either directing the diffusion trajectory (such as physical tunnels) or limiting the diffusion space (such as covalent tethering). , In other words, to achieve substrate channeling, interactions usually need to be introduced between the enzyme complexes and the intermediates (cofactors, for instance), which maintain colocalization of the intermediates with the enzymes and prevent the intermediates from diffusing into the bulk solution. Therefore, the design and construction of biocatalytic materials capable of cofactor channeling requires suitable platforms, where functionally coupled enzymes and their cofactors can be colocalized in a controlled manner.…”

“…12−14 To further enhance the utilization−regeneration process of cofactor at low quantity, substrate channeling has been proposed in the design of biocatalytic materials. 8,15 Compared to conventional multienzyme cascades (no channeling), inducing channeling of a cofactor requires the directed transfer of the cofactor between an enzyme couple without its diffusion into the bulk solution, which can potentially increase the effective local concentration of the cofactor available to the enzymes. As a result, even with a very small input quantity of cofactor, the system can still operate at high catalytic rates.…”