Enhancing Multistep Reactions: Biomimetic Design of Substrate Channeling Using P22 Virus-Like Particles

Abstract

Many biocatalytic processes inside cells employ substrate channeling to control the diffusion of intermediates for improved efficiency of enzymatic cascade reactions. This inspirational mechanism offers a strategy for increasing efficiency of multistep biocatalysis, especially where the intermediates are expensive cofactors that require continuous regeneration. However, it is challenging to achieve substrate channeling artificially in vitro due to fast diffusion of small molecules. By mimicking some naturally occurring metabolons, nanoreactors are developed using P22 virus-like particles (VLPs), which enhance the efficiency of nicotinamide adenine dinucleotide (NAD)-dependent multistep biocatalysis by substrate channeling. In this design, NAD-dependent enzyme partners are coencapsulated inside the VLPs, while the cofactor is covalently tethered to the capsid interior through swing arms. The crowded environment inside the VLPs induces colocalization of the enzymes and the immobilized NAD, which shuttles between the enzymes for in situ regeneration without diffusing into the bulk solution. The modularity of the nanoreactors allows to tune their composition and consequently their overall activity, and also remodel them for different reactions by altering enzyme partners. Given the plasticity and versatility, P22 VLPs possess great potential for developing functional materials capable of multistep biotransformations with advantageous properties, including enhanced efficiency and economical usage of enzyme cofactors.

Keywords: biomimetic materials; cascade reactions; cofactor regeneration; modular nanoreactors; multistep catalysis; substrate channeling; virus-like particles.

Figure 1

Bioinspiration for and biomimetic design of P22 VLP nanoreactors capable of an enhanced multistep reaction. a) i) Carboxysome catalyzes the two‐step carbon fixation. Multiple copies of carbonic anhydrase (CA) and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) are colocalized inside the protein compartment (left; this is only a model representation and does not reflect the actual scenario in carboxysome; PDB: 7CKC, 7ZBT, 2FGY). Due to selective permeability of the capsid, CO₂ produced by CA is prevented from diffusing out of the capsid, which increases the local concentration of the intermediate close to RuBisCO and induces channeling (right). ii) Acetyl‐CoA carboxylase catalyzes a two‐step carboxyl transfer reaction. The cofactor, biotin, is covalently attached to biotin carboxyl carrier protein (BCCP) domain by a swing arm and shuttled between biotin carboxylase (BC) domain and carboxylase transferase (CT) domain, which induces channeling of the reaction intermediate. b) In this biomimetic nanoreactor, two functionally coupled enzymes, PtDH and AdhD, are encapsulated inside the P22 VLP, and their cofactor, NAD, is covalently tethered to the VLP interior by a swing arm. P22 VLP provides a colocalization environment while the swing arm limits the diffusion of the cofactor intermediate. c) Covalent immobilization of NAD is realized by bioconjugation between engineered P22 VLP and NAD‐maleimide (the reduced form is shown in the structure). d) The biomimetic P22 nanoreactor can be realized by in vitro assembly.

Figure 2

Activity of the biomimetic P22 VLP nanoreactors. a) A linear scheme of the two‐step hydride transfer reaction. Phosphate is depicted as the final product. b) The length of the swing arm is longer in i) NAD‐CP_N‐ext than ii) NAD‐CP_S39C, which likely influences the accessibility of the immobilized NAD to the encapsulated enzymes. c) Kinetic progression of phosphate production for i) CP_N‐ext particles and ii) CP_S39C particles (mean ± s.e.m., n = 3). The early stage (0–60 min) of the reaction is zoomed in. The rate of the steady state phase is estimated by the average velocity between 90 and 180 min, displayed in the histograms.

Figure 3

Tuning the composition of the VLP nanoreactors. a) i) The quantity of immobilized NAD can be tuned by mixing NAD‐CP_N‐ext and unlabeled CP_N‐ext during in vitro assembly. ii) SDS‐PAGE shows the percentage of NAD‐CP_N‐ext is successfully altered, where the percentage is estimated by densitometry analysis (Figure S14, Supporting Information). iii) VLP nanoreactors with more NAD loading exhibits higher kinetic efficiency of the catalysis of the two‐step reaction, determined by phosphate production (mean ± s.e.m., n = 3). b) i) The enzyme ratio in the nanoreactors can be tuned by altering enzyme stoichiometry during in vitro assembly. ii) SDS‐PAGE shows enzyme ratio is successfully altered among different assemblies, where the AdhD:PtDH ratio is estimated by densitometry analysis (Figure S17, Supporting Information). iii) The enzyme ratio affects the multistep activity of the nanoreactors, determined by phosphate production (mean ± s.e.m., n = 3).

Figure 4

The biomimetic P22 VLP nanoreactors as functional and modular biocatalytic materials. a) i) The production of 2,3‐butanediol is investigated as the final product of the two‐step reaction. ii) The NAD‐CP_N‐ext particles showed continuous biocatalytic activity for 24 h (mean ± s.e.m., n = 3). b) i) NAD‐CP_N‐ext can be recycled from in vitro assembled particles and the unassembled proteins during in vitro assembly, for remodeling the nanoreactors for another two‐step hydride transfer reaction catalyzed by PtDH and an ene reductase (ER). ii) SDS‐PAGE shows that incubation with Ni²⁺ chelate resin removed most of the His‐tagged enzymes from NAD‐CP_N‐ext (I = input, i.e., proteins before incubation with His‐tag affinity resin; B = resin‐bound fraction; U = unbound fraction; U_c = concentrated unbound fraction; U and U_c were used for the new round of in vitro assembly). iii) The new nanoreactors are active in catalysis of the reduction of carbon–carbon double bond in cyclohexenone (mean ± s.e.m., n = 3).