Characterization and functional validation of glyoxalase II from rice

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Abstract

Glyoxalase II, one of the enzymes of the glyoxalase pathway, cDNA cloned from rice (OsglyII) consists of 1623 nucleotides with an open reading frame of 1010 bp encoding a polypeptide of 336 amino acids and an estimated isoelectric point of 8.08. The recombinant protein purified from Escherichia coli using Ni–NTA affinity chromatography showed molecular mass of ∼37 kDa. Catalytic parameters of the protein were determined using S-d-lactoylglutathione as a thioester substrate. The Km (61 μM) and Kcat (301 s−1) values were lower than those reported for Arabidopsis, human and yeast and showed pH optima at 7.2. The E. coli overexpressing OsglyII were able to grow on higher concentration of methylglyoxal. Transcript analysis in rice showed that OsglyII gene expression is stimulated within 15 min in response to various abiotic stresses as well as treatment with abscisic acid or salicylic acid. This multistress response of OsglyII gene documents its future utility in developing tolerance to various stresses in crop plants.

Section snippets

Cloning and sequence analysis of OsglyII

The rice glyII cDNA (OsglyII) was cloned by PCR based approach from rice cDNA library [30]. Sequence analysis revealed one full length cDNA clone of OsglyII (Accession No. AY054407) based on homology with Accession No. U90927 [23]. For homology analysis, BLAST searches were conducted using GenBank. GlyII protein sequences were aligned using the ClustalW multiple alignment program (MacVector). Dendrogram was prepared from amino acid sequences between various glyII sequences already reported in

Molecular cloning and sequence comparison of OsglyII cDNA with other glyII in the data bank

The sequence analysis of the cloned gene encoding for glyII enzyme of glyoxalase pathway from rice (OsglyII) revealed that the cDNA clone is 1623 bp long with an open reading frame of 1010 bp (representing the full length glyII coding sequence) and a 108 bp long 5′ UTR and 504 bp long 3′ UTR. The deduced amino acid sequence length for glyII was found to be 336 residues. The estimated molecular mass of the protein was ∼37 kDa with an isoelectric point of 8.08. The glyII from Arabidopsis and spinach

Acknowledgments

We thank Professor Ray Wu, Cornell University, USA for valuable suggestions and critical reading of the manuscript. Thanks are also due to Drs. F. White and B.W. Porter, Kansas State University, USA, for the initial glyoxalase II clone. The financial support by the Department of Biotechnology (DBT, New Delhi) Rice Network Project, International Foundation for Science, Sweden research grant to SLS-P, DBT Post-Doc fellowship to S.K.Y. and grants from the International Centre for Genetic

References (39)

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1

Present address: Biotechnology Division, Institute of Himalayan Bioresource Technology, Palampur 176061, India.

2

Both the authors have contributed equally to this work.

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