Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA.
A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K(m) was determined to be 120 microM using S-d-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA.
Saxena
Mukesh
M
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Bisht
Rekha
R
Roy
Suchandra Deb
SD
Sopory
S K
SK
Bhalla-Sarin
Neera
N
D000040:Abscisic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009149:Mustard Plant / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology; D013869:Thiolester Hydrolases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation; D015032:Zinc / Q000494:pharmacology; D000099301:Hydroxyacylglutathione Hydrolase
D000040:Abscisic Acid; D024101:Mitochondrial Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D013869:Thiolester Hydrolases; D015032:Zinc; D000099301:Hydroxyacylglutathione Hydrolase