FUNCTIONAL EXPRESSION OF A TOBACCO GENE-RELATED TO THE SERINE HYDROLASE FAMILY - ESTERASE-ACTIVITY TOWARDS SHORT-CHAIN DINITROPHENYL ACYLESTERS

We have recently reported the isolation of a tobacco gene, hsr-203J, w hose transcripts accumulate during the hypersensitive reaction, a plan t response associated with resistance to pathogens. We present and dis cuss here some structural and biochemical properties of the gene produ ct. Nucleotide sequence analysis has shown that the hsr 203J gene cont ains an open reading frame coding for a polypeptide of 335 amino acids . The predicted amino acid sequence contains the GXSXG motif character istic of serine hydrolases, and displays limited but significant simil arity to lipases and esterases of prokaryotic origin. The hsr 203J gen e was expressed in Escherichia coli, and the recombinant protein, puri fied to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hyd rolyze lipids, and was active on short-chain acyl esters only. The hyd rolytic activity was abolished by diisopropyl fluorophosphate and a de rivative of isocoumarin, as expected for a member of the serine hydrol ase family. Sequence similarities between the tobacco esterase and exp ressed sequence tags ill databases suggest the existence of members of this enzyme family in various plant species.