A heartwood norlignan, (E)-hinokiresinol, is formed from 4-coumaryl 4-coumarate by a Cryptomeria japonica enzyme preparation

An enzyme preparation from the cultured cells of Cryptomeria japonica catalyses the formation of a heartwood norlignan, (E)-hinokiresinol, from two distinct phenylpropanoid monomers: 4-coumaroyl CoA and 4-coumaryl alcohol, and from a dimer: 4-coumaryl 4-coumarate.

Norlignans, which have the 1,3-diarylpentane structure, are found in coniferous trees (especially heartwood) and some monocotyledonous plants, including Asparagus officinalis.1,2

Their biosynthesis is especially of interest in relation to heartwood formation, a metabolic event specific to woody plants.1–3

Hinokiresinol is the simplest of the norlignans and therefore a good target for norlignan biosynthetic studies.

Hinokiresinol has two geometrical (E)- and (Z)-isomers.

(E)-Hinokiresinol is the source of the heartwood colour for Chamaecyparis obtusa (hinoki cypress),4 and is typically found in conifer heartwood together with other norlignans,5–8 in contrast to the distribution of (Z)-hinokiresinol in monocotyledonous herbs including A. officinalis.2,3

We have demonstrated for the first time the biosynthetic process and enzymatic formation of (Z)-hinokiresinol using the A. officinalis cell system (Scheme 1), the formation of 4-coumaryl 4-coumarate by the coupling of 4-coumaryl alcohol and 4-coumaroyl CoA, and the subsequent rearrangement to yield (Z)-hinokiresinol.2,3

Our attention was next directed to the biosynthesis of (E)-hinokiresinol in relation to heartwood formation.

Herein, we report for the first time, enzymatic (E)-hinokiresinol formation using the Cryptomeria japonica (Japanese cedar) cell system as an enzyme source.

Callus was initiated from the shoot tips excised aseptically from stems of C. japonica cv.

Kumotooshi (collected at Wakayama University Forest, Kyoto University, in November 2002) on Murashige and Skoog (MS) gel medium (0.3% phytagel, 2% sucrose, 1 × 10−3 g l−1 of 2,4-dichlorophenoxyacetic acid, and 1 × 10−4 g l−1 of kinetin).

A cell-suspension culture was obtained by transferring callus crumps to the MS liquid medium: 1% sucrose, 0.7 g l−1 of KCl, 0.93 g l−1 of KNO3, free of NH4NO3, 1 × 10−3 g l−1 of 2,4-dichlorophenoxyacetic acid, and 1 × 10−4 g l−1 of kinetin.

The culture was maintained by continuous shaking (80 rpm) at 25 °C in the dark.

The cells were maintained for over one year by repeated subculturing every 10 days.

Cell-free extracts were prepared as described previously.3

Briefly, the cells incubated for 10 days after the last subculturing were frozen with liquid nitrogen and pulverized with a mortar and pestle.

The powder obtained was ground in the presence of polyclar AT (30% of cell weight), sea sand, and 100 mM Tris-HCl buffer (THB) (pH 7.8) containing 15 mM 2-mercaptoethanol (ME), and the resultant slurry was filtered with gauze.

The filtrate obtained was centrifuged, and the supernatant was applied to a Sephadex G-25 column, which had been pre-equilibrated and was eluted with 20 mM THB (pH 7.8) containing 15 mM ME.

To the excluded fraction was added ammonium sulfate (0–70% saturation), and the resulting precipitates were recovered by centrifugation and desalted by passing through a Sephadex G-25 column as above.

The effluent was used as the enzyme preparation.

Two distinct phenylpropanoid monomers, 4-[7,9,9-2H3]coumaryl alcohol and 4-coumaroyl CoA3 were incubated with the enzyme preparation at 30 °C (Table 1, A-1) and then the product was analysed by GC-MS as previously described.3

The mass spectrum (Fig. 1A) is that of authentic (Z)-hinokiresinol TMS ether showing principal ions at m/z 396 (molecular ion, [M]+) and 230.

Although the mass spectrum is quite similar to that of (E)-hinokiresinol TMS ether (Fig. 1B), the (Z)- and (E)-isomers are distinguishable from each other by retention times (tR), as shown in Fig. 2A.

Fig. 1C is the spectrum of the (E)-hinokiresinol fraction obtained following incubation of 4-[7,9,9-2H3]coumaryl alcohol and 4-coumaroyl CoA with the enzyme preparation, and its tR is shown in Fig. 2B.

As can be seen in Fig 1C, in addition to the ions at m/z 396 and 230 which are derived from the unlabelled internal standard, ions at m/z 399 ([M]+ + 3) and 233 are found, indicating the formation of (E)-[2H3]hinokiresinol.

The slightly smaller tR in the selected ion monitoring (SIM) chromatogram of m/z 399 of the fraction (16.54 min, Fig. 2B) compared to that of molecular ion (m/z 396) of unlabelled (E)-hinokiresinol (16.57 min, Fig. 2A), which is ascribed to the well-known isotope effect on GC retention time, further supports the formation of the deuterium labelled (E)-hinokiresinol.

Proof that the formation of (E)-[2H3]hinokiresinol was enzymatic was obtained by control experiments (Table 1); the formation of (E)-[2H3]hinokiresinol from 4-[7,9,9-2H3]coumaryl alcohol and 4-coumaroyl CoA was not observed when the denatured enzyme preparation was used, and when the enzyme preparation or the substrate(s) were omitted from the complete assay.

Next we incubated (7E,7′E)-4-[7′,9′,9′-2H3]coumaryl 4-coumarate3 with the enzyme preparation at 30 °C.

Like the result observed with the conversion of 4-[7,9,9-2H3]coumaryl alcohol and 4-coumaroyl CoA, (7E,7′E)-4-[7′,9′,9′-2H3]coumaryl 4-coumarate was found to be efficiently converted to (E)-[2H3]hinokiresinol (Figs. 1D and 2C, and Table 1, B-1).

To eliminate the involvement of (Z)-hinokiresinol as an intermediate in the formation of (E)-hinokiresinol, we incubated (E)-hinokiresinol and (Z)-hinokiresinol individually with the enzyme preparation.

GC-MS analysis showed that neither (Z)- nor (E)-hinokiresinol was converted to the corresponding geometrical isomers under the conditions employed (data not shown), indicating that (E)-hinokiresinol was directly formed from 4-coumaryl 4-coumarate with the enzyme preparation, but not via (Z)-hinokiresinol.

Taken together, our results have provided evidence for the (E)-hinokiresinol synthesizing system, which does not involve (Z)-hinokiresinol as an intermediate.

In addition, the present results, together with our previous report,3 indicate that there are at least two hinokiresinol synthesizing systems in plants; one giving rise to the (E)-isomer, the other to the (Z)-isomer.

They are probably two distinct hinokiresinol synthases, namely (E)- and (Z)-hinokiresinol synthases catalysing the direct conversion of (7E,7′E)-4-coumaryl 4-coumarate to (E)-hinokiresinol and (Z)-hinokiresinol, respectively.

In fact, we have demonstrated that a purified Asparagus enzyme transformed (7E,7′E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol (unpublished).

However, it is still possible that (7E,7′E)-4-coumaryl 4-coumarate was first isomerised to (7Z,7′E)-4-coumaryl 4-coumarate which was then transformed to give rise to (E)-hinokiresinol by the Cryptomeria crude enzyme preparation.

In conclusion, the present study has demonstrated for the first time enzyme preparation catalysed (E)-hinokiresinol formation and this is the first report of the enzymatic formation of a heartwood norlignan.

Parts of this research were supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no.

12660150) and by TOSTEM Foundation for Construction Materials Industry Promotion, and performed as one of the technology development projects of the “Green Biotechnology Program” supported by the New Energy and Industrial Technology Department Organization.

The authors are indebted to the Field Science Education and Research Center, Kyoto University for supplying Cryptomeria japonica cv.


Thanks are also due to Bob and Dori Downs for their valuable comments on English writing, and Miyuki Nakamura for her technical assistance in callus culture maintenance.