Crystal Structures of Complexes of the Branched-Chain Aminotransferase from Deinococcus radiodurans with α-Ketoisocaproate and l- Glutamate Suggest the Radiation Resistance of This Enzyme for Catalysis

Branched-chain aminotransferases (BCAT), which utilize pyridoxal 5′-phosphate (PLP) as a cofactor, reversibly catalyze the transfer of the α-amino groups of three of the most hydrophobic branched-chain amino acids (BCAA), leucine, isoleucine, and valine, to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. The BCAT fromDeinococcus radiodurans (Dr BCAT), an extremophile, was cloned and expressed inEscherichia coli for structure and functional studies. The crystal structures of the nativeDr BCAT with PLP and its complexes withl -glutamate and α-ketoisocaproate (KIC), respectively, have been determined. TheDr BCAT monomer, comprising 358 amino acids, contains large and small domains connected with an interdomain loop. The cofactor PLP is located at the bottom of the active site pocket between two domains and near the dimer interface. The substrate (l -glutamate or KIC) is bound with key residues through interactions of the hydrogen bond and the salt bridge near PLP inside the active site pocket. Mutations of some interaction residues, such as Tyr71, Arg145, and Lys202, result in loss of the specific activity of the enzymes. In the interdomain loop, a dynamic loop (Gly173 to Gly179) clearly exhibits open and close conformations in structures ofDr BCAT without and with substrates, respectively.Dr BCAT shows the highest specific activity both in nature and under ionizing radiation, but with lower thermal stability above 60°C, than either BCAT fromEscherichia coli (e BCAT) or fromThermus thermophilus (HB8BCAT). The dimeric molecular packing and the distribution of cysteine residues at the active site and the molecular surface might explain the resistance to radiation but small thermal stability ofDr BCAT.