Pinus taeda phenylpropenal double-bond reductase: purification, cDNA cloning, heterologous expression in Escherichia coli, and subcellular localization in P. taeda.
A phenylpropenal double-bond reductase (PPDBR) was obtained from cell suspension cultures of loblolly pine (Pinus taeda L.). Following trypsin digestion and amino acid sequencing, the cDNA encoding this protein was subsequently cloned, with the functional recombinant protein expressed in Escherichia coli and characterized. PPDBR readily converted both dehydrodiconiferyl and coniferyl aldehydes into dihydrodehydrodiconiferyl and dihydroconiferyl aldehydes, when NADPH was added as cofactor. However, it was unable to reduce directly either the double bond of dehydrodiconiferyl or coniferyl alcohols in the presence of NADPH. During this reductive step, the corresponding 4-proR hydrogen was abstracted from [4R-3H]-NADPH during hydride transfer. This is thus the first report of a double-bond reductase involved in phenylpropanoid metabolism, and which is presumed to be involved in plant defense. In situ mRNA hybridization indicated that the PPDBR transcripts in P. taeda stem sections were localized to the vascular cambium, as well as to radial and axial parenchyma cell types. Additionally, using P. taeda cell suspension culture crude protein extracts, dehydrodiconiferyl and coniferyl alcohols could be dehydrogenated to afford dehydrodiconiferyl and coniferyl aldehydes. Furthermore, these same extracts were able to convert dihydrodehydrodiconiferyl and dihydroconiferyl aldehydes into the corresponding alcohols. Taken together, these results indicate that in the crude extracts dehydrodiconiferyl and coniferyl alcohols can be converted to dihydrodehydrodiconiferyl and dihydroconiferyl alcohols through a three-step process, i.e. by initial phenylpropenol oxidation, then sequential PPDBR and phenylpropanal reductions, respectively.
Kasahara
Hiroyuki
H
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Jiao
Ying
Y
Bedgar
Diana L
DL
Kim
Sung-Jin
SJ
Patten
Ann M
AM
Xia
Zhi-Qiang
ZQ
Davin
Laurence B
LB
Lewis
Norman G
NG
D000449:Aldehyde Reductase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D028223:Pinus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013347:Subcellular Fractions / Q000201:enzymology
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D000449:Aldehyde Reductase