Identifying a Cinnamoyl Coenzyme A Reductase (CCR) Activity with 4-Coumaric Acid: Coenzyme A Ligase (4CL) Reaction Products in Populus tomentosa

다국어 초록 (Multilingual Abstract)

A cinnamoyl coenzyme A reductase (CCR, EC

1.2.1.44), one of the key enzyme involved in lignin

biosynthesis, was cloned from Populus tomentosa (Chinese

white poplar). At the same time, a 4CL1 gene was cloned

from P. tomentosa, too. The two genes were subcloned in

pQE31 vector and expressed in Escherichia coli M15. Both

of them were purified by Ni-NTA. Purified CCR protein

was digested by trypsin and analyzed by HPLC-MS; the

peptide segments had 27% similarity with the sequence of

the CCR protein. 4CL was thought to be a neighbor

enzyme of CCR in lignin biosynthesis. In this paper, a

4CL1 from P. tomentosa was cloned, and its enzyme

reaction products were extracted for the substrates of CCR.

Three 4CL1 enzyme reaction products were monitored by

HPLC-MS and then the CCR enzyme reaction was detected

by GC-MS. In the CCR reaction, the three corresponding

aldehyde (p-coumaraldehyde, caffealdehyde, and coniferaldehyde)

were detected and identified by Frontier3

software. The results showed that the CCR that we cloned

from P. tomentosa had affinities with 4CL1 enzyme

reaction products and a ptCCR that was cloned from aspen

(Li et al., Plant Cell Physiol 46(7):1073–1082, 2005) only

had affinity with feruloyl-CoA. The different results maybe

depend on the different study method. The method of

exacting 4CL enzyme products as the substrates of CCR in

the paper was reliable and can be used in lignin

biosynthesis network to detect the enzymes in the neighborhood

that depended on the polarity of the substrates and

products. This CCR gene had eight homology sequence

CCR gene when a BLAST was conducted in Populus

trichocarpa genome database. The CCR homology genes in

Populus suggested that some CCRs may take part in the

lignin biosynthesis, too. The gene family would be the hot

spot in the future study.

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