Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants.

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

Next generation sequencing in predicting gene function in podophyllotoxin biosynthesis.

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

The laccase multigene family in Arabidopsis thaliana: towards addressing the mystery of their gene function(s).

While laccases, multi-copper glycoprotein oxidases, are often able to catalyze oxidation of a broad range of substrates, such as phenols and amines in vitro, their precise physiological/biochemical roles in higher plants remain largely unclear, e.g., Arabidopsis thaliana contains 17 laccases with only 1 having a known physiological function. To begin to explore their roles in planta, spatial and temporal expression patterns of Arabidopsis laccases were compared and contrasted in different tissues at various development stages using RT-PCR and promoter-GUS fusions. Various cell-specific expressions were noted where specific laccases were uniquely expressed, such as LAC4 in interfascicular fibers and seed coat columella, LAC7 in hydathodes and root hairs, LAC8 in pollen grains and phloem, and LAC15 in seed coat cell walls. Such specific cell-type expression patterns provide new leads and/or strategies into determining their precise physiological/biochemical roles. In addition, there was an apparent redundancy of gene expression patterns for several laccases across a wide variety of tissues, lignified and non-lignified, perhaps indicative of overlapping function(s). Preliminary evidence, based on bioinformatics analyses, suggests that most laccases may also be tightly regulated at both transcriptional (antisense transcripts, histone and DNA methylation) and posttranscriptional (microRNAs) levels of gene expression.

Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans.

(-)-Matairesinol is a central biosynthetic intermediate to numerous 8-8'-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos. It is formed by action of an enantiospecific secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative "mammalian" lignan or "phytoestrogen" enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 angstrom resolution, respectively. The enzyme is a homotetramer, consisting of an alpha/beta single domain monomer containing seven parallel beta-strands flanked by eight alpha-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD(+) and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.