Isupov MN et al. (2019), Thermostable Branched-Chain Amino Acid Transami...

ECB-ART-46942

Front Bioeng Biotechnol 2019 Jan 01;7:7. doi: 10.3389/fbioe.2019.00007.

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

Isupov MN , Boyko KM , Sutter JM , James P , Sayer C , Schmidt M , Schönheit P , Nikolaeva AY , Stekhanova TN , Mardanov AV , Ravin NV , Bezsudnova EY , Popov VO , Littlechild JA .

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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.

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Genes referenced: LOC100893907 LOC115925415 LOC579470

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References [+] :

Altschul, Basic local alignment search tool. 1990, Pubmed

	Scheme 1. Reaction of Branched Chain TAms.
	Figure 1. Electron density for the bound substrate AKG in the active site of subunit A of the GEO1900. The 2Fo-Fc electron density map (blue) is contoured at 1.5σ, the positive (green) and negative (red) Fo-Fc electron density maps are contoured at 3.5σ and −3.5σ, respectively. Figure drawn using PyMOL (Schrödinger, LLC).
	Figure 2. A cartoon representation of the subunit of GEO1900 with the large domain shown in gold and the small domain shown in ice blue. The loops connecting the domains are shown in coral. The cofactor PLP and the active site lysine 152 are shown as stick models with carbon atoms shown in green. Figures 2–5, 7, 8 are drawn using CCP4mg (McNicholas et al., 2011).
	Figure 3. A cartoon representation of the dimer of the AF0933 BCAT gabaculine complex. The gabaculine-PLP covalent adduct molecules are shown as spheres.
	Figure 4. A cartoon representation of the AF0933 BCAT holo structure in the native hexameric form. The PLP cofactor molecules are shown as spheres.
	Figure 5. A view from outside onto the active site cavity entrance using a surface representation of the AF0933 BCAT complex with the inhibitor gabaculine (shown as spheres).
	Figure 6. Multiple sequence alignment of different BCATs, A. flugidus, G. acetivorans, T. thermophilus, E.coli, T. uzoniensis, N. haematococca. Arrows indicate β-strands, and helical curves denote α-helices of the structure of AF0933 above and Nectria TAm below. The active site lysine is highlighted in red. The figure was prepared with ESPript3 (Robert and Gouet, 2014).
	Figure 7. An overlay of the Cα trace of the AF0933 BCAT (green and coral coil) gabaculine complex with the Nectria TAm (ice blue and gold coil) gabaculine complex (Sayer et al., 2014) to illustrate the differences between the ligand and loop conformations within the active site between the two enzymes. The PLP gabaculine adduct is shown as a stick model for the AF0933 BCAT structure and as thick lines for the Nectria amine TAm structure. Interdomain loops and the loop connecting β5 and β6 of the N-terminal small domain of the adjacent subunit in the catalytic dimer covering the active site cavity, have different conformation between the AF0933 BCAT and the Nectria TAm as shown.
	Figure 8. A cartoon representation of the superimposition of the catalytic dimers of the AF0933 gabaculine complex with the Nectria TAm gabaculine complex, illustrating the additional α-helix at the N-terminal region of Nectria TAm as shown in Figure 6. The PLP gabaculine adduct is shown in ball and stick mode.