Tomoyuki Nakatsubo

Characterization of Arabidopsis thaliana Pinoresinol Reductase, a New Type of Enzyme Involved in Lignan Biosynthesis

A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (–)-pinoresinol to (–)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (–)-pinoresinols to (+)- and (–)-lariciresinols efficiently with comparative kcat/Km values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.

Metabolic analysis of the cinnamate/monolignol pathway in Carthamus tinctorius seeds by a stable-isotope-dilution method

The present study established a system for comprehensive metabolic analysis of the cinnamate/monolignol and lignan pathways by the use of a stable-isotope-dilution method. The system was successfully applied to characterization of the pathways in Carthamus tinctorius cv. Round-leaved White maturing seeds in combination with administration of stable-isotope-labelled precursors. Experimental results obtained using this technique strongly suggested the intermediacy of ferulic acid in lignan biosynthesis in the plant.

At5g54160 gene encodes Arabidopsis thaliana 5-hydroxyconiferaldehyde O-methyltransferase

The function of an Arabidopsis thaliana gene, At5g54160 annotated as a caffeic acid O-methyltransferase CAOMT gene was characterized. The recombinant enzyme of this gene (AtOMT1) catalyzed the O-methylation of phenylpropanoid and flavonoid substrates. The specificity constants (kcat/Km) for 5-hydroxyconiferaldehyde (5-HCAld) and quercetin were both 0.11 μM−1·min−1. On the other hand, lignins of At5g54160-knockout Arabidopsis mutants lacked syringyl units. In addition, we showed that the gene silencing also resulted in significant accumulation of caffeyl alcohol (CaAlc). These results strongly suggested that At5g54160 gene is involved in syringyl lignin synthesis for the methylation of both 5-hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compound(s).

First in vitro norlignan formation with Asparagus officinalis enzyme preparation

We report for the first time that an enzyme preparation from fungal-elicited Asparagus officinalis cultured cells catalyses the formation of a norlignan, (Z)-hinokiresinol, from two non-identical phenylpropanoid monomers, 4-coumaryl alcohol and 4-coumaroyl CoA, and from a dimer, 4-coumaryl 4-coumarate, without any additional cofactors.

The subunit composition of hinokiresinol synthase controls geometrical selectivity in norlignan formation

The selective formation of E- or Z-isomers is an important process in natural product metabolism. We show that the subunit composition of an enzyme can alter the geometrical composition of the enzymatic products. Hinokiresinol synthase, purified from Asparagus officinalis cell cultures, is responsible for the conversion of (7E,7′E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol, the first step in norlignan formation. The protein is most likely a heterodimer composed of two distinct subunits, which share identity with members of the phloem protein 2 gene superfamily. Interestingly, each recombinant subunit of hinokiresinol synthase expressed in Escherichia coli solely converted (7E,7′E)-4-coumaryl 4-coumarate to the unnatural (E)-hinokiresinol, the E-isomer of (Z)-hinokiresinol. By contrast, a mixture of recombinant subunits catalyzed the formation of (Z)-hinokiresinol from the same substrate.

Stereoselectivity of the Biosynthesis of Norlignans and Related Compounds

Norlignans, lignans, and neolignans are biosynthesized by coupling two units of phenylpropanoid monomers including p-hydroxycinnamyl alcohols and allyl- and propenylphenols. Each molecule of the phenylpropanoid dimers is usually chiral, and naturally occurring norlignans, lignans, and neolignans are often optically active. The subunit compositions of norlignan synthase (hinokiresinol synthase) are known to control enantiomeric selectivity as well as cis/trans selectivity during norlignan formation. This contrasts sharply with the dirigent protein-mediated enantioselective control in lignan biosynthesis. Indeed, the dirigent protein is a unique asymmetric inducer that plays prominent roles in enantioselective lignan biosynthesis. Recently, however, enzymes that are involved in the subsequent metabolic steps, such as pinoresinol (/lariciresinol) reductase, were also found to play significant roles in the enantioselective formation of lignans. In this review, recent advances in the biosynthesis of norlignans, lignans, and neolignans are discussed in relation to enantioselective control mechanisms.