DNA–Nanoparticle Composites Synergistically Enhance Organophosphate Hydrolase Enzymatic Activity

Cell-free synthetic biology relies on optimally exploiting enzymatic activity, and recent demonstrations that nanoparticle (NP) and DNA scaffolding can enhance enzyme activity suggest new avenues toward this. A modular architecture consisting of a DNA cage displaying semiconductor quantum dots (QDs) that, in turn, ratiometrically display the organophosphate hydrolase phosphotriesterase (PTE) was utilized as a model system. Increasing DNA cage concentration relative to QD-PTE and creating a dense composite enhanced PTE rates up to 12.5-fold, suggesting strong synergy between the NP and DNA components; this putatively arises from increased enzymatic stability and alleviation of its rate-limiting step. Such bioinorganic composites may offer new scaffolding approaches for synthetic biology

Enhancement of functional PTE production by QDs.

(A) Reaction setup highlighting stopping of the CFPS reactions with kanamycin at different time points. Paraoxon hydrolysis tracked by measurement of the p-nitrophenol absorbance product. Schematic not to scale. (B) PURExpress® reaction with QDs produced functional PTE, the activity of which was monitored by absorbance. Kanamycin was added at various time points to quench translation. (C) Identical PURExpress® reaction without QDs treated in the same manner as panel (B) produced less functional PTE, resulting in less activity and p-nitrophenol product absorbance. PTE PDB ID: IPTA [76]. Other protein structures are the same as shown in Fig 1.</p

Schematic depicting enhanced cell-free protein synthesis from aggregating the intrinsic enzymes around NPs.

(CFPS) systems can suffer from limited reaction rates, likely due to diffusion between components as shown in the reaction to left. CdSe/ZnS core/shell quantum dots (QDs) bind the His-tag of some CFPS components and cross-link into NP-aggregates to bring them into proximity, potentially increasing the catalytic rates or product yield. The enzyme structures shown are known to be present in the CFPS utilized and are represented by structures drawn from the PDB. IDs: 1CRK (mitochondrial creatine kinase), 1FMT (E. coli methionyl-tRNA(f)Met formyltransferase), and, 3PCO (E. coli phenylalanine-tRNA synthetase) [56–60].</p

Exploration of the In Vitro Violacein Synthetic Pathway with Substrate Analogues

Evolution has gifted enzymes with the ability to synthesize an abundance of small molecules with incredible control over efficiency and selectivity. Central to an enzyme’s role is the ability to selectively catalyze reactions in the milieu of chemicals within a cell. However, for chemists it is often desirable to extend the substrate scope of reactions to produce analogue(s) of a desired product and therefore some degree of enzyme promiscuity is often desired. Herein, we examine this dichotomy in the context of the violacein biosynthetic pathway. Importantly, we chose to interrogate this pathway with tryptophan analogues in vitro, to mitigate possible interference from cellular components and endogenous tryptophan. A total of nine tryptophan analogues were screened for by analyzing the substrate promiscuity of the initial enzyme, VioA, and compared to the substrate tryptophan. These results suggested that for VioA, substitutions at either the 2- or 4-position of tryptophan were not viable. The seven analogues that showed successful substrate conversion by VioA were then applied to the five enzyme cascade (VioABEDC) for the production of violacein, where l-tryptophan and 6-fluoro-l-tryptophan were the only substrates which were successfully converted to the corresponding violacein derivative(s). However, many of the other tryptophan analogues did convert to various substituted intermediaries. Overall, our results show substrate promiscuity with the initial enzyme, VioA, but much less for the full pathway. This work demonstrates the complexity involved when attempting to analyze substrate analogues within multienzymatic cascades, where each enzyme involved within the cascade possesses its own inherent promiscuity, which must be compatible with the remaining enzymes in the cascade for successful formation of a desired product

sfGFP production enhancement with full range of QD concentrations tested at 0.5x PURExpress® reaction conditions.

(A) Production of sfGFP fluorescence in arbitrary units over time versus that of the “free” or QD negative reaction. Samples were excited at 485 nm and fluorescence monitored at 510 nm [69]. (TIF)</p

Additional statistics of sfGFF production at 0.5x PURExpress® reaction conditions.

ANOVA p-value was F was > Fcrit, indicating significant difference between treatments. Tukey-Kramer analysis was then done. Stars indicate treatments were significantly different from each other (alpha 0.05). (TIF)</p

sfGFP production enhancement with full range of QD concentrations tested at 1x PURExpress® reaction conditions.

(A) Production of sfGFP fluorescence in arbitrary units over time versus that of the “free” or QD negative reaction. Samples were excited at 485 nm and fluorescence monitored at 510 nm [69]. (TIF)</p

Raw images of all gels and blots.

(TIF)</p

Method for additional statistical analysis of sfGFP production.

(DOCX)</p

Inhibition of cell-free reaction by kanamycin.

Indicated concentration of kanamycin was added after 30 min in cell-free reaction and change in sfGFP fluorescence was monitored for 90 min. Initial time point is the time of kanamycin addition. (TIF)</p