MDPI - Publisher of Open Access Journals

13 pages, 1265 KiB

Open AccessArticle

Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles

by Scott N. Dean, Meghna Thakur, Joseph R. Spangler, Aaron D. Smith, Sean P. Garin, Scott A. Walper and Gregory A. Ellis

Bioengineering 2023, 10(5), 583; https://doi.org/10.3390/bioengineering10050583 - 11 May 2023

Cited by 9 | Viewed by 2712

Abstract

All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered E. coli to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein “anchors/directors”) and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp’, SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp’. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp’ anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs. Full article

(This article belongs to the Special Issue Development and Biomedical Applications of Bacterial Carriers and Bacteria-Derived Biomimetic Vectors)

► Show Figures

Graphical abstract

15 pages, 2572 KiB

Open AccessArticle

Probing the Suitability of Different Ca²⁺ Parameters for Long Simulations of Diisopropyl Fluorophosphatase

by Alexander Zlobin, Igor Diankin, Sergey Pushkarev and Andrey Golovin

Molecules 2021, 26(19), 5839; https://doi.org/10.3390/molecules26195839 - 26 Sep 2021

Cited by 7 | Viewed by 2937

Abstract

Organophosphate hydrolases are promising as potential biotherapeutic agents to treat poisoning with pesticides or nerve gases. However, these enzymes often need to be further engineered in order to become useful in practice. One example of such enhancement is the alteration of enantioselectivity of diisopropyl fluorophosphatase (DFPase). Molecular modeling techniques offer a unique opportunity to address this task rationally by providing a physical description of the substrate-binding process. However, DFPase is a metalloenzyme, and correct modeling of metal cations is a challenging task generally coming with a tradeoff between simulation speed and accuracy. Here, we probe several molecular mechanical parameter combinations for their ability to empower long simulations needed to achieve a quantitative description of substrate binding. We demonstrate that a combination of the Amber19sb force field with the recently developed 12-6 Ca²⁺ models allows us to both correctly model DFPase and obtain new insights into the DFP binding process. Full article

(This article belongs to the Special Issue Application of Computational Methods and Biomolecular Structural Modeling to the Investigation of Metalloproteins)

► Show Figures

Figure 1

18 pages, 7859 KiB

Open AccessReview

Enzymatic Degradation of Organophosphorus Pesticides and Nerve Agents by EC: 3.1.8.2

by Marek Matula, Tomas Kucera, Ondrej Soukup and Jaroslav Pejchal

Catalysts 2020, 10(12), 1365; https://doi.org/10.3390/catal10121365 - 24 Nov 2020

Cited by 16 | Viewed by 4654

Abstract

The organophosphorus substances, including pesticides and nerve agents (NAs), represent highly toxic compounds. Standard decontamination procedures place a heavy burden on the environment. Given their continued utilization or existence, considerable efforts are being made to develop environmentally friendly methods of decontamination and medical countermeasures against their intoxication. Enzymes can offer both environmental and medical applications. One of the most promising enzymes cleaving organophosphorus compounds is the enzyme with enzyme commission number (EC): 3.1.8.2, called diisopropyl fluorophosphatase (DFPase) or organophosphorus acid anhydrolase from Loligo Vulgaris or Alteromonas sp. JD6.5, respectively. Structure, mechanisms of action and substrate profiles are described for both enzymes. Wild-type (WT) enzymes have a catalytic activity against organophosphorus compounds, including G-type nerve agents. Their stereochemical preference aims their activity towards less toxic enantiomers of the chiral phosphorus center found in most chemical warfare agents. Site-direct mutagenesis has systematically improved the active site of the enzyme. These efforts have resulted in the improvement of catalytic activity and have led to the identification of variants that are more effective at detoxifying both G-type and V-type nerve agents. Some of these variants have become part of commercially available decontamination mixtures. Full article

► Show Figures

Graphical abstract

12 pages, 7239 KiB

Open AccessArticle

Theoretical Studies on Catalysis Mechanisms of Serum Paraoxonase 1 and Phosphotriesterase Diisopropyl Fluorophosphatase Suggest the Alteration of Substrate Preference from Paraoxonase to DFP

by Hao Zhang, Ling Yang, Ying-Ying Ma, Chaoyuan Zhu, Shenghsien Lin and Rong-Zhen Liao

Molecules 2018, 23(7), 1660; https://doi.org/10.3390/molecules23071660 - 7 Jul 2018

Cited by 13 | Viewed by 4141

Abstract

The calcium-dependent β-propeller proteins mammalian serum paraoxonase 1 (PON1) and phosphotriesterase diisopropyl fluorophosphatase (DFPase) catalyze the hydrolysis of organophosphorus compounds and enhance hydrolysis of various nerve agents. In the present work, the phosphotriesterase activity development between PON1 and DFPase was investigated by using the hybrid density functional theory method B3LYP. Based on the active-site difference between PON1 and DFPase, both the wild type and the mutant (a water molecule replacing Asn270 in PON1) models were designed. The results indicated that the substitution of a water molecule for Asn270 in PON1 had little effect on the enzyme activity in kinetics, while being more efficient in thermodynamics, which is essential for DFP hydrolysis. Structure comparisons of evolutionarily related enzymes show that the mutation of Asn270 leads to the catalytic Ca²⁺ ion indirectly connecting the buried structural Ca²⁺ ion via hydrogen bonds in DFPase. It can reduce the plasticity of enzymatic structure, and possibly change the substrate preference from paraoxon to DFP, which implies an evolutionary transition from mono- to dinuclear catalytic centers. Our studies shed light on the investigation of enzyme catalysis mechanism from an evolutionary perspective. Full article

(This article belongs to the Special Issue Theoretical Investigations of Reaction Mechanisms)

► Show Figures

Figure 1

18 pages, 4373 KiB

Open AccessArticle

Theoretical Studies Applied to the Evaluation of the DFPase Bioremediation Potential against Chemical Warfare Agents Intoxication

by Flávia V. Soares, Alexandre A. De Castro, Ander F. Pereira, Daniel H. S. Leal, Daiana T. Mancini, Ondrej Krejcar, Teodorico C. Ramalho, Elaine F. F. Da Cunha and Kamil Kuca

Int. J. Mol. Sci. 2018, 19(4), 1257; https://doi.org/10.3390/ijms19041257 - 23 Apr 2018

Cited by 22 | Viewed by 5229

Abstract

Organophosphorus compounds (OP) are part of a group of compounds that may be hazardous to health. They are called neurotoxic agents because of their action on the nervous system, inhibiting the acetylcholinesterase (AChE) enzyme and resulting in a cholinergic crisis. Their high toxicity and rapid action lead to irreversible damage to the nervous system, drawing attention to developing new treatment methods. The diisopropyl fluorophosphatase (DFPase) enzyme has been considered as a potent biocatalyst for the hydrolysis of toxic OP and has potential for bioremediation of this kind of intoxication. In order to investigate the degradation process of the nerve agents Tabun, Cyclosarin and Soman through the wild-type DFPase, and taking into account their stereochemistry, theoretical studies were carried out. The intermolecular interaction energy and other parameters obtained from the molecular docking calculations were used to construct a data matrix, which were posteriorly treated by statistical analyzes of chemometrics, using the PCA (Principal Components Analysis) multivariate analysis. The analyzed parameters seem to be quite important for the reaction mechanisms simulation (QM/MM). Our findings showed that the wild-type DFPase enzyme is stereoselective in hydrolysis, showing promising results for the catalytic degradation of the neurotoxic agents under study, with the degradation mechanism performed through two proposed pathways. Full article

(This article belongs to the Special Issue Computational Studies of Structure-Dynamics-Function Relationships in Biomolecules)

► Show Figures

Graphical abstract

Search Results (5)

Further Information

Guidelines

MDPI Initiatives

Follow MDPI

Saved Queries

Search Filter Reset All

Years

Feature Papers

Subjects

Journals

Article Types

Countries / Regions

Search Results (5)

Further Information

Guidelines

MDPI Initiatives

Follow MDPI