Computational redesign of transaminase active site

Aminotransferases are widely exploited in simple as well as more elaborate multi-enzymatic cascade reactions as an environmentally friendly alternative to transition metal catalysis. However, efficient selective conversion of numerous targets is a great limitation to date [1]. Attempts to improve substrate scope have been undertaken by generation and screening of large mutant libraries, which is very time-consuming and raises costs concerns [2]. Recent approaches explored the use of molecular docking of demanding substrates, followed by energy minimization and/or MD simulations [1;3]. Still, the best results have been obtained by extensive mutagenesis and screening. Please click Additional Files below to see the full abstract

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

The biodegradation of the nylon-6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP-dependent hydrolytic ring-opening to 6-aminohexanoate. This non-natural ω-amino acid is converted to 6-oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I PLP enzymes. To understand the structural basis of 6-aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat /KM ) with the caprolactam degradation intermediates 6-aminohexanoate and 6-oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three-dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6-aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate-binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6-aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6-aminohexanoate degradation. This article is protected by copyright. All rights reserved

Robust ω-Transaminases by Computational Stabilization of the Subunit Interface

Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from Pseudomonas jessenii. A large number of surface-located point mutations and mutations predicted to stabilize the subunit interface were examined. Experimental screening revealed that 10 surface mutations out of 172 tested were indeed stabilizing (6% success), whereas testing 34 interface mutations gave 19 hits (56% success). Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for stability. After mutations were combined, 2 very stable variants with 4 and 6 mutations were obtained, which in comparison to wild type (Tm app = 62 °C) displayed Tm app values of 80 and 85 °C, respectively. These two variants were also 5-fold more active at their optimum temperatures and tolerated high concentrations of isopropylamine and cosolvents. This allowed conversion of 100 mM acetophenone to (S)-1-phenylethylamine (>99% enantiomeric excess) with high yield (92%, in comparison to 24% with the wild-type transaminase). Crystal structures mostly confirmed the expected structural changes and revealed that the most stabilizing mutation, I154V, featured a rarely described stabilization mechanism: namely, removal of steric strain. The results show that computational interface redesign can be a rapid and powerful strategy for transaminase stabilization

Non-Functional Jaw Muscular Activity in Patients with Disorders of Consciousness Revealed by A Long-Lasting Polygraphy

The presence of involuntary, non-functional jaw muscle activity (NFJMA) has not yet been assessed in patients with disorders of consciousness (DOC), although the presence of bruxism and other forms of movement disorders involving facial muscles is probably more frequent than believed. In this work, we evaluated twenty-two prolonged or chronic DOC patients with a long-lasting polygraphic recording to verify NFJMA occurrence and assess its neurophysiological patterns in this group of patients. A total of 5 out of 22 patients showed the presence of significant NFJMA with electromyographic patterns similar to what can be observed in non-DOC patients with bruxism, thus suggesting a disinhibition of masticatory motor nuclei from the cortical control. On the other hand, in two DOC patients, electromyographic patterns advised for the presence of myorhythmia, thus suggesting a brainstem/diencephalic involvement. Functional, non-invasive tools such as long-lasting polygraphic recordings should be extended to a larger sample of patients, since they are increasingly important in revealing disorders potentially severe and impacting the quality of life of DOC patients

L'espace domestique au Bronze final et au premier âge du Fer dans le sud de la Corse

We present a synthetic approach to the kinds of housing found in the mountainous Alta Rocca sector of southern Corsica during the late Bronze Age and Early Iron Age. Study of protohistoric non-fortified sites in Corsica is relatively new, but the numerous projects carried out in recent years reveal the structural and developmental complexity of living spaces, thus permitting an initial comparative analysis. This is based mainly on the excavations of the large settlement of Cuciurpula that began in 2008 and are still in progress. It also makes use of the 2012 test pits in the villages of Puzzonu and Nuciaresa. The chronology of the areas that were studied cover the entire time span from the 12th to the 4th century B.C., and thus permit one to understand the developmental changes that occurred with respect to both the overall settlement organization and the internal space and architecture of the dwellings