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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.

Structural Studies

The GEO1900 holoform (internal aldimine form; GEO1900_holo) and the unproductive complex of holoenzyme with the keto substrate AKG-(GEO1900_AKG) have been determined to 1.9 and 2.2 Å resolution, respectively, in the trigonal space group P3221. There are three GEO1900 subunits per asymmetric unit, which form a tight hexamer with 32 point group symmetry with their symmetry mates. The AF0933 structure has been determined in the holoform (AF0933_holo) and in complex with the inhibitor gabaculine (GABC) (AF0933_GABC) to 2.1 and 2.0 Å resolution, respectively, in the orthorhombic P212121 space group. The asymmetric unit contains a hexameric AF0933 molecule which is very similar to GEO1900 hexamer observed in the crystal. All of the structures have been refined to low R-factors with good stereochemical parameters as shown in Table . In the GEO1900 structures two N-terminal amino acids which are not present in AF0933, were not defined in the electron density. The interdomain loop residues 121–129 could be fully built into the subunits A and B in GEO1900_AKG structure. The bound ligand AKG was found only in subunit A and was clearly defined in the electron density (Figure ). The interdomain loop could only be fully built in one subunit (D) out of the six subunits in both the AF0933_holo and AF0933_GABC structures. The crystal contacts in AF0933 appear to be contributing to the lower disorder of the loop in the D subunit. The inhibitor, gabaculine was not present in the electron density in the subunit F of AF0933, therefore this subunit was built as an internal aldimine in the AF0933_GABC structure. In other subunits the gabaculine-PLP adduct was modeled with partial occupancy in the range of 0.6–0.7. There are no residues in a cis conformation in the well-defined regions of the GEO1900 and AF0933 structures. Residue Glu249 is a Ramachandran plot outlier (Ramakrishnan and Ramachandran, ) in all subunits of both AF0933 structures. The corresponding Glu251 has similar main chain angles in the GEO1900 structures, but lies in a generously allowed region of the Ramachandran plot (Laskowski et al., ). The fungal class IV TAms from Aspergillus and Nectria species (Łyskowski et al., ; Sayer et al., ; Thomsen et al., ) have a Gly residue at this position. Many residues in both of the GEO1900 and AF0933 structures have been modeled with multiple conformations of some amino acid side chains. The residues Gln107 and Asn108 were modeled with alternative conformations of the main chain in most subunits of the AF0933_holo structure and in the subunits E and F of the AF0933_GABC structure.