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, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens. We present and discuss here some structural and biochemical properties of the gene product. Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids. The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin. The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hydrolyze lipids, and was active on short-chain acyl esters only. The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family. Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.
Baudouin
E
E
Laboratoire Signaux et Messages Cellulaires chez les Végétaux, UMR 5546 CNRS, Université Paul Sabatier, Toulouse, France.
Charpenteau
M
M
Roby
D
D
Marco
Y
Y
Ranjeva
R
R
Ranty
B
B
D002087:Butyrates / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D007531:Isoflurophate / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010947:Plants, Toxic; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D002087:Butyrates; D004791:Enzyme Inhibitors; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007531:Isoflurophate; C033592:4-nitrophenyl butyrate; D004950:Esterases; C088007:hsr203J protein, Nicotiana tabacum; D002265:Carboxylic Ester Hydrolases