A Plant-specific Tau Class Glutathione S-transferase from Oryza sativa Having Significant Detoxification Activity Towards Chloroacetanilide Herbicides

Abstract

Glutathione S-transferases (GSTs, EC 2.5.1.18) are enzymes that detoxify endobiotic and xenobiotic compounds by covalently linking glutathione (GSH) to a hydrophobic substrate, forming a less reactive and more polar glutathione S-conjugate. 1 In plants, GSTs detoxify herbicides, organic pollutants, and natural toxins; they also protect cells from a wide range of biotic and abiotic stresses, including pathogen attack, xenobiotic and heavy-metal toxicity, and oxidative stress. 2,3 Plant GSTs are grouped into five classes (phi, zeta, tau, theta and lambda) based on sequence identity, gene organization, and active site residues in the proteins. A further GST-like class, DHAR (proteins with dehydroascorbate reductase activity), was recently reported with members in Arabidopsis, rice and soybean. 2 The function of these plant-specific GSTs is detoxification of herbicides in both crops and weeds. Despite all available research, both the biochemical properties and functions of plant GSTs remain to be elucidated. Rice is the most important crop for human consumption with more than a half of the world’s population utilizing it as an energy source. The entire rice genomes for the japonica and indica subspecies have been sequenced. 4 From these genome sequences, a GST gene homolog (GenBank Accession No. AF309379) was identified from Oryza sativa by homolog searches in the NCBI database. As yet, there have been no reports concerning this gene product. Therefore, we cloned this gene and expressed it in Escherichia coli. The mRNA from Oryza sativa L. cv. Yamahousi was isolated from the cell culture and transcribed into cDNA. Primers for PCR, using the cDNA as a template, were derived from a published cDNA sequence from Oryza sativa L. cv. Japonica. 5 Subsequently, a 702-bp fragment of Oryza sativa cDNA was amplified by PCR, subcloned into the expression vector pET-26b(+) and transformed into the E. coli strain BL21 (DE3). Both DNA strands were sequenced with the final amino acid sequence bearing a difference between the PCR-identified sequence and the earlier cDNAderived sequence (GenBank Accession No. AF309379). The gene whereby guanine was substituted with thymine at position of nucleotide 166 gave an Asn 55 residue instead of Lys 55. The cloned gene was composed of 702-bp encoding for the 233 amino acids and designated OsGSTU3. A multiple alignment of the OsGSTU3 protein with other GSTs is shown in Figure 1. Sequence aliment was performed using the T-Coffee and Boxshade programe (http:// www.ch.embnet.org). GST isoenzymes belonging to the same class showed more than approximately 60% identity in their primary structure, whereas enzymes belonging to phi and theta classes generally had less than 2% sequence identity. 2 Zeta class enzymes also bore less than 24% sequence identity. Based on its gene sequence, OsGSTU3 was presumed to belong to the plant-specific tau class GST. The E. coli BL21 (pET-OsGSTU3) clone was tested for production of recombinant OsGSTU3 protein. The expressed OsGSTU3 protein was purified by S-hexylGSH affinity