Engineering of haloalkane dehalogenase enantioselectivity towards βbromoalkanes: Open-solvated versus occluded-desolvated active sites

Enzymatic catalysis is widely used for preparing optically pure chemicals. Natural catalysts have to be often optimized to exhibit sufficient enantioselectivity towards industrially attractive non-natural substrates. Understanding the molecular basis of enzyme–substrate interactions involved in enantiodiscrimination is essential for rational design of selective catalysts. Haloalkane dehalogenases (EC 3.8.1.5) can convert a broad range of halogenated aliphatic compounds to their corresponding alcohols via SN2 mechanism [1]. The very first haloalkane dehalogenase exhibiting high enantioselectivity towards β-brominated alkanes (E-values of up to 174) was DbjA from Bradyrhizobium japonicum USDA110 [2]. This enzyme has a wide open solvent-accessible active site and its enantioselectivity towards β-brominated alkanes is modulated by a surface loop unique to DbjA [2]. Assuming that the active site geometry is crucial for substrate recognition, it was proposed that DbjA’s enantioselectivity could be transferred to closely related, but non-selective DhaA from Rhodococcus rhodochrous NCIMB13064 [1] by active site transplantation [3]. The unique loop fragment from DbjA together with additional 8-point substitutions was inserted to DhaA. Although the crystal structure of resulting variant DhaA12 exhibited identical geometry of the active site and the access tunnel as DbjA, it did not reach identical level of hydration and flexibility and lacked enantioselectivity towards β-bromoalkanes (E-value = 18) [3]. Interestingly, the variant DhaA31 constructed independently with a goal to enhance enzyme activity towards anthropogenic compound 1,2,3-trichlopropane [4], exhibited high enantioselectivity towards 2-bromopentane (E-value = 179) [5] as DbjA (E-value = 174) [2, 3]. DhaA31 contains five mutations, I135F, C176Y, V245F, L246I and Y273F, located in a main and a slot tunnel. Four of five mutations are large and aromatic residues narrowing two access tunnels and occluding the enzyme active site [4]. The level of DhaA31 active site hydration, so important for DbjA’s enantioselectivity [2, 3] is low, suggesting a different structural basis of enantioselectivity towards 2-bromopentane. A systematic study on the molecular basis of enantioselectivity in DbjA, DhaA, and DhaA31 using thermodynamic and kinetic analyses, site-directed mutagenesis, and molecular modeling was carried out. DhaA31 enantioselectivity arises from the hydrophobic substrate’s interactions with the occluded and desolvated active site [5], while DbjA enantioselectivity results from water-mediated interactions of 2-bromopentane with the active site’s hydrophobic wall [2]. Our data imply that enantioselectivity of haloalkane dehalogenases can be achieved by both occluded-desolvated active site and open-solvated active site. The engineering of “DbjA-like” enantioselectivity by modification of the active site hydration remains challenging. References: 1. Koudelakova, T., et al. 2013. Biotechnol. J. 8: 32–45. Prokop, Z., et al. 2010. Angew. Chem. Int. Ed., 49: 6111-6115. Sykora, J., et al. 2014. Nat. Chem. Biol., 10: 428-430. Pavlova, M., et al. 2009. Nat. Chem. Biol., 5: 727-733. Liskova, V., et al. 2017. Angew. Chem. Int. Ed., DOI: 10.1002/anie.201611193

New Model of Ventral Spinal Cord Lesion Induced by Balloon Compression in Rats.

Despite the variety of experimental models of spinal cord injury (SCI) currently used, the model of the ventral compression cord injury, which is commonly seen in humans, is very limited. Ventral balloon compression injury reflects the common anatomical mechanism of a human lesion and has the advantage of grading the injury severity by controlling the inflated volume of the balloon. In this study, ventral compression of the SCI was performed by the anterior epidural placement of the balloon of a 2F Fogarty's catheter, via laminectomy, at the level of T10. The balloon was rapidly inflated with 10 or 15 μL of saline and rested in situ for 5 min. The severity of the lesion was assessed by behavioral and immunohistochemical tests. Compression with the volume of 15 μL resulted in severe motor and sensory deficits represented by the complete inability to move across a horizontal ladder, a final Basso, Beattie and Bresnahan (BBB) score of 7.4 and a decreased withdrawal time in the plantar test (11.6 s). Histology and immunohistochemistry revealed a significant loss of white and gray matter with a loss of motoneuron, and an increased size of astrogliosis. An inflation volume of 10 μL resulted in a mild transient deficit. There are no other balloon compression models of ventral spinal cord injury. This study provided and validated a novel, easily replicable model of the ventral compression SCI, introduced by an inflated balloon of Fogarty´s catheter. For a severe incomplete deficit, an inflated volume should be maintained at 15 μL

Computer-assisted stabilization of fibroblast growth factor FGF-18

The fibroblast growth factors (FGF) family holds significant potential for addressing chronic diseases. Specifically, recombinant FGF18 shows promise in treating osteoarthritis by stimulating cartilage formation. However, recent phase 2 clinical trial results of sprifermin (recombinant FGF18) indicate insufficient efficacy. Leveraging our expertise in rational protein engineering, we conducted a study to enhance the stability of FGF18. As a result, we obtained a stabilized variant called FGF18-E4, which exhibited improved stability with 16 °C higher melting temperature, resistance to trypsin and a 2.5-fold increase in production yields. Moreover, the FGF18-E4 maintained mitogenic activity after 1-week incubation at 37 °C and 1-day at 50 °C. Additionally, the inserted mutations did not affect its binding to the fibroblast growth factor receptors, making FGF18-E4 a promising candidate for advancing FGF-based osteoarthritis treatment

Strategies for Stabilization of Enzymes in Organic Solvents

One of the major barriers to the use of enzymes in industrial biotechnology is their insufficient stability under processing conditions. The use of organic solvent systems instead of aqueous media for enzymatic reactions offers numerous advantages, such as increased solubility of hydrophobic substrates or suppression of water-dependent side reactions. For example, reverse hydrolysis reactions that form esters from acids and alcohols become thermodynamically favorable. However, organic solvents often inactivate enzymes. Industry and academia have devoted considerable effort into developing effective strategies to enhance the lifetime of enzymes in the presence of organic solvents. The strategies can be grouped into three main categories: (i) isolation of novel enzymes functioning under extreme conditions, (ii) modification of enzyme structures to increase their resistance toward nonconventional media, and (iii) modification of the solvent environment to decrease its denaturing effect on enzymes. Here we discuss successful examples representing each of these categories and summarize their advantages and disadvantages. Finally, we highlight some potential future research directions in the field, such as investigation of novel nanomaterials for immobilization, wider application of computational tools for semirational prediction of stabilizing mutations, knowledge-driven modification of key structural elements learned from successfully engineered proteins, and replacement of volatile organic solvents by ionic liquids and deep eutectic solvents

The Role of Green Tea Catechin Epigallocatechin Gallate (EGCG) and Mammalian Target of Rapamycin (mTOR) Inhibitor PP242 (Torkinib) in the Treatment of Spinal Cord Injury

Spinal cord injury (SCI) is a devastating condition that has physical and psychological consequences for patients. SCI is accompanied by scar formation and systemic inflammatory response leading to an intense degree of functional loss. The catechin, epigallocatechin gallate (EGCG), an active compound found in green tea, holds neuroprotective features and is known for its anti-inflammatory potential. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two functionally distinct complexes termed mTOR complex 1 and 2 (mTORC1; mTORC2). Inhibition of mTORC1 by rapamycin causes neuroprotection, leading to partial recovery from SCI. In this study the effects of EGCG, PP242 (an inhibitor of both complexes of mTOR), and a combination of EGCG and PP242 in SCI have been examined. It has been found that both EGCG and PP242 significantly improved sensory/motor functions following SCI. However, EGCG appeared to be more effective (BBB motor test, from 2 to 8 weeks after SCI, p = 0.019, p = 0.007, p = 0.006, p = 0.006, p = 0.05, p = 0.006, and p = 0.003, respectively). The only exception was the Von Frey test, where EGCG was ineffective, while mTOR inhibition by PP242, as well as PP242 in combination with EGCG, significantly reduced withdrawal latency starting from week three (combinatorial therapy (EGCG + PP242) vs. control at 3, 5, and 7 weeks, p = 0.011, p = 0.007, and p = 0.05, respectively). It has been found that EGCG was as effective as PP242 in suppressing mTOR signaling pathways, as evidenced by a reduction in phosphorylated S6 expression (PP242 (t-test, p t-test, p = 0.0002)). These results demonstrate that EGCG and PP242 effectively suppress mTOR pathways, resulting in recovery from SCI in rats, and that EGCG acts via suppressing mTOR pathways