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

Chung De Chen, Chih Hao Lin, Phimonphan Chuankhayan, Yen Chieh Huang, Yin Cheng Hsieh, Tien Feng Huang, Hong Hsiang Guan, Ming Yih Liu, Wen Chang Chang, Chun Jung Chena

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Abstract

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 from Deinococcus radiodurans (DrBCAT), an extremophile, was cloned and expressed in Escherichia coli for structure and functional studies. The crystal structures of the native DrBCAT with PLP and its complexes with L-glutamate and α-ketoisocaproate (KIC), respectively, have been determined. The DrBCAT 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 of DrBCAT without and with substrates, respectively. DrBCAT shows the highest specific activity both in nature and under ionizing radiation, but with lower thermal stability above 60oC, than either BCAT from Escherichia coli (eBCAT) or from Thermus 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 of DrBCAT.

Original languageEnglish
Pages (from-to)6206-6216
Number of pages11
JournalJournal of Bacteriology
Volume194
Issue number22
DOIs
Publication statusPublished - Nov 1 2012
Externally publishedYes

Fingerprint

Deinococcus
Pyridoxal Phosphate
Catalysis
Glutamic Acid
Radiation
Catalytic Domain
Enzymes
Hot Temperature
Escherichia coli
Thermus thermophilus
Keto Acids
Branched Chain Amino Acids
Isoleucine
Valine
Ionizing Radiation
Leucine
Cysteine
Hydrogen
Salts
Amino Acids

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

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. / Chen, Chung De; Lin, Chih Hao; Chuankhayan, Phimonphan; Huang, Yen Chieh; Hsieh, Yin Cheng; Huang, Tien Feng; Guan, Hong Hsiang; Liu, Ming Yih; Chang, Wen Chang; Chena, Chun Jung.

In: Journal of Bacteriology, Vol. 194, No. 22, 01.11.2012, p. 6206-6216.

Research output: Contribution to journalArticle

Chen, Chung De ; Lin, Chih Hao ; Chuankhayan, Phimonphan ; Huang, Yen Chieh ; Hsieh, Yin Cheng ; Huang, Tien Feng ; Guan, Hong Hsiang ; Liu, Ming Yih ; Chang, Wen Chang ; Chena, Chun Jung. / 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. In: Journal of Bacteriology. 2012 ; Vol. 194, No. 22. pp. 6206-6216.
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abstract = "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 from Deinococcus radiodurans (DrBCAT), an extremophile, was cloned and expressed in Escherichia coli for structure and functional studies. The crystal structures of the native DrBCAT with PLP and its complexes with L-glutamate and α-ketoisocaproate (KIC), respectively, have been determined. The DrBCAT 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 of DrBCAT without and with substrates, respectively. DrBCAT shows the highest specific activity both in nature and under ionizing radiation, but with lower thermal stability above 60oC, than either BCAT from Escherichia coli (eBCAT) or from Thermus 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 of DrBCAT.",
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AU - Lin, Chih Hao

AU - Chuankhayan, Phimonphan

AU - Huang, Yen Chieh

AU - Hsieh, Yin Cheng

AU - Huang, Tien Feng

AU - Guan, Hong Hsiang

AU - Liu, Ming Yih

AU - Chang, Wen Chang

AU - Chena, Chun Jung

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