Thermostable Branched-Chain Amino Acid Transaminases From the Archaea Geoglobus acetivorans and Archaeoglobus fulgidus: Biochemical and Structural Characterization

Abstract

Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, Geoglobus acetivorans, and Archaeoglobus fulgidus. These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in Escherichia coli and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to (R)-methylbenzylamine but no activity to (S)-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the Geoglobus enzyme and 2.0 Å for the Archaeoglobus enzyme. Also the Geoglobus enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the Archaeoglobus enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another (R) selective class IV transaminase from the fungus Nectria haematococca which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.

An (R)-Selective Transaminase From Thermomyces stellatus: Stabilizing the Tetrameric Form

“…Other highly similar RTAs, including At RTA (4ce5, 81% identity) (Łyskowski et al, 2014 ), and RTAs from Nectria haematococca (4cmd, 78% identity) (Sayer et al, 2014 ), Exophiala xenobiotica (6fte, 70% identity) (Telzerow et al, 2019 ), and Aspergillus fumigatus (4uug, 73% identity) (Thomsen et al, 2014 ) are reported as dimers based on their crystal packing. Two thermostable BCATs, belonging to the same fold type IV as RTAs, with hexameric structures have recently been reported by Isupov et al ( 2019 ). In addition, increased operational stability of tetrameric STAs compared to dimeric STAs has been reported (Börner et al, 2017 ).…”

Exploration of Bioengineered Scaffolds Composed of Thermo-Responsive Polymers for Drug Delivery in Wound Healing

“…Fused Deposition Modeling (FDM) is being explored for processing polymers into drug delivery systems required by special populations for treating rare diseases. The process consists of a plastic filament that is pushed into a heating head ( Figure 4 ) [ 110 ]. The contact causes the filament to soften into a semi-solid state that forms the sequential layers by extrusion.…”

Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases

“…In addition to the commercially available enzymes, many groups have modified TA enzymes using rational and semirational protein engineering and directed evolution methods [90]. More thermally stable and robust TA enzymes of the groups 3 and 4 have been identi fied and characterised from thermophilic genomes and metagenomes [91,92].…”