Dopamine D1 vs D5 receptor-dependent induction of seizures in relation to DARPP-32, ERK1/2 and GluR1-AMPA signalling.

Recent reports have shown that the selective dopamine D(1)-like agonist SKF 83822 [which stimulates adenylate cyclase, but not phospholipase C] induces prominent behavioral seizures in mice, whereas its benzazepine congener SKF 83959 [which stimulates phospholipase C, but not adenylate cyclase] does not. To investigate the relative involvement of D(1) vs D(5) receptors in mediating seizures, ethological behavioral topography and cortical EEGs were recorded in D(1), D(5) and DARPP-32 knockout mice in response to a convulsant dose of SKF 83822. SKF 83822-induced behavioral and EEG seizures were gene dose-dependently abolished in D(1) knockouts. In both heterozygous and homozygous D(5) knockouts, the latency to first seizure was significantly increased and total EEG seizures were reduced relative to wild-types. The majority (60%) of homozygous DARPP-32 knockouts did not have seizures; of those having seizures (40%), the latency to first seizure was significantly increased and the number of high amplitude, high frequency polyspike EEG events was reduced. In addition, immunoblotting was performed to investigate downstream intracellular signalling mechanisms at D(1)-like receptors following challenge with SKF 83822 and SKF 83959. In wild-types administered SKF 83822, levels of ERK1/2 and GluR1 AMPA receptor phosphorylation increased two-fold in both the striatum and hippocampus; in striatal slices DARPP-32 phosphorylation at Thr34 increased five-fold relative to vehicle-treated controls. These findings indicate that D(1), and to a lesser extent D(5), receptor coupling to DARPP-32, ERK1/2 and glutamatergic signalling is involved in mediating the convulsant effects of SKF 83822

Expanding the Toolbox of (R)-selective Amine Transaminases by Identification and Characterization of new Members

Telzerow A, Paris J, Hakansson M, et al. Expanding the Toolbox of (R)-selective Amine Transaminases by Identification and Characterization of new Members. Chembiochem : a European journal of chemical biology. 2020.Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. (R) -selective ATAs belong to the fold type IV PLP dependent enzymes and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R) -ATAs with additional bias towards fungal origin. We aimed to expand the toolbox of (R) -ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R) -ATAs. The ATA from Exophiala sideris contains a motif characteristic for D-ATAs, which was previously believed to be a disqualifying factor for (R) -ATA activity. The crystal structure of the ATA from Shinella is the first from a gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R) -ATAs with a shortened/missing N-terminal helix. The active site charges vary significantly between the new and known ATAs correlating with their diverging substrate scope. © 2020 Wiley-VCH GmbH

Amine Transaminase from Exophiala Xenobiotica-Crystal Structure and Engineering of a Fold IV Transaminase that Naturally Converts Biaryl Ketones

Telzerow A, Paris J, Hakansson M, et al. Amine Transaminase from Exophiala Xenobiotica-Crystal Structure and Engineering of a Fold IV Transaminase that Naturally Converts Biaryl Ketones. ACS CATALYSIS. 2019;9(2):1140-1148.Amine transaminases are frequently used for the production of chiral amines starting from prochiral ketones. These amines can be applied as active pharmaceutical ingredients or drug precursors. However, there are still limitations to the use of amine transaminases when it comes to bulky ketone substrates, such as biaryl ketones. Using data mining, an (R)-selective amine transaminase from Exophiala xenobiotica was identified which naturally converts biaryl ketone substrates to the corresponding amines with up to 85% conversion and excellent enantioselectivity (>99% ee). Its protein crystal structure was obtained with a resolution of 1.52 angstrom, which enabled us to explain this interesting substrate acceptance. Structure-guided protein engineering resulted in a quintuple variant with increased stability. Moreover, the amino acid exchange T273S increased the activity and broadened the substrate scope, enabling conversions of various biaryl ketones with up to >99%. A preparative biotransformation of 1-(4-(pyridin-3-yl)phenyl)ethenone at 75 mM (15 g/L) resulted in 96% of isolated yield of the respective amine

The cross-reactive calcium-binding pollen allergen, Phl p 7, reveals a novel dimer assembly

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients