PMID- 12392563
OWN - NLM
STAT- MEDLINE
DCOM- 20021213
LR  - 20190620
IS  - 0014-2956 (Print)
IS  - 0014-2956 (Linking)
VI  - 269
IP  - 21
DP  - 2002 Nov
TI  - Brassica napus soluble epoxide hydrolase (BNSEH1).
PG  - 5295-302
AB  - Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy 
      fatty acids and in mediating defence responses. We report the cloning of a 
      full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) 
      obtained by screening of a cDNA library prepared from methyl jasmonate induced 
      leaf tissue, and the 5'-RACE technique. The cDNA encodes a soluble protein 
      containing 318 amino acid residues. The identity on the protein level is 85% to 
      an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from 
      other plants. A 5 x His tag was added to the N-terminus of the BNSEH1 and the 
      construct was over-expressed in the yeast Pichia pastoris. The recombinant 
      protein was recovered at high levels after Ni-agarose chromatography of lysed 
      cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on 
      Western blots with antibodies raised to a sEH from Arabidopsis thaliana. BNSEH1 
      was shown to be a monomer by gel filtration analysis. The activity was low 
      towards cis-stilbene oxide but much higher using trans-stilbene oxide as 
      substrate with Vmax of 0.47 micro mol.min.mg-1, Km of 11 micro m and kcat of 0.3 
      s-1. The optimum temperature of the recombinant enzyme was 55 degrees C and the 
      optimum pH 6-7 for trans-stilbene oxide hydrolysis. The isolation of BNSEH1 will 
      facilitate metabolic engineering of epoxy fatty acid metabolism for functional 
      studies of resistance and seed oil modification in this important oilcrop.
FAU - Bellevik, Stefan
AU  - Bellevik S
AD  - Department of Plant Biology, Genetics Center, Swedish University of Agricultural 
      Sciences, Uppsala, Sweden. Stefan.Bellevik@vbiol.slu.se
FAU - Zhang, Jiaming
AU  - Zhang J
FAU - Meijer, Johan
AU  - Meijer J
LA  - eng
SI  - GENBANK/AJ459780
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Eur J Biochem
JT  - European journal of biochemistry
JID - 0107600
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Plant Proteins)
RN  - 0 (Recombinant Proteins)
RN  - EC 3.3.2.- (Epoxide Hydrolases)
SB  - IM
MH  - Amino Acid Sequence
MH  - Brassica napus/*enzymology
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Enzyme Activation/drug effects/physiology
MH  - Enzyme Inhibitors/pharmacology
MH  - Epoxide Hydrolases/biosynthesis/*chemistry/*genetics
MH  - Hydrogen-Ion Concentration
MH  - Molecular Sequence Data
MH  - Molecular Weight
MH  - Pichia/genetics
MH  - Plant Proteins/biosynthesis/chemistry/genetics
MH  - Recombinant Proteins/biosynthesis/chemistry/genetics
MH  - Sequence Homology, Amino Acid
MH  - Solubility
MH  - Temperature
EDAT- 2002/10/24 04:00
MHDA- 2002/12/17 04:00
CRDT- 2002/10/24 04:00
PHST- 2002/10/24 04:00 [pubmed]
PHST- 2002/12/17 04:00 [medline]
PHST- 2002/10/24 04:00 [entrez]
AID - 3247 [pii]
AID - 10.1046/j.1432-1033.2002.03247.x [doi]
PST - ppublish
SO  - Eur J Biochem. 2002 Nov;269(21):5295-302. doi: 10.1046/j.1432-1033.2002.03247.x.