Table_1_Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges.docx

Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.</p

Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature

Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid Loligo vulgaris and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 °C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications

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

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

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

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

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

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

Estimated characteristics of PURExpress® components.

(XLSX)</p