Accelerated degradation of lignin by lignin peroxidase isozyme H8 (LiPH8) from Phanerochaete chrysosporium with engineered 4-O-methyltransferase from Clarkia breweri.

Free-hydroxyl phenolic units can decrease or even abort the catalytic activity of lignin peroxidase H8 during oxidation of veratryl alcohol and model lignin dimers, resulting in slow and inefficient lignin degradation. In this study we applied engineered 4-O-methyltransferase from Clarkia breweri to detoxify the inhibiting free-hydroxyl phenolic groups by converting them to methylated phenolic groups. The multistep, enzyme-catalyzed process that combines 4-O-methyltransferase and lignin peroxidase H8 suggested in this work can increase the efficiency of lignin-degradation. This study also suggests approaching the field of multi-enzyme in vitro systems to improve the understanding and development of plant biomass in biorefinery operations.

Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves.

Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used.

Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase catalytic domain in Saccharomyces cerevisiae.

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T(73) was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.

Essential region for 3-N methylation in N-methyltransferases involved in caffeine biosynthesis.

The caffeine biosynthetic pathway is composed of three methylation steps, and N-methyltransferase catalyzing each step has high substrate specificity. Since the amino acid sequences among coffee 7-methylxanthosine synthase (CmXRS1), theobromine synthase, and caffeine synthase are highly homologous to each other, these substrate specificities seem to be determined in a very restricted region. The analysis of site-directed mutants for CmXRS1 that naturally acts at the initial step, i.e., 7-N methylation of xanthosine, revealed that the activity of 3-N methylation needs a histidine residue at corresponding position 161 in the CmXRS1 sequence. We succeeded in producing the mutant enzyme which can catalyze the first and second methylation steps in caffeine biosynthesis.

Engineering monolignol 4-O-methyltransferases to modulate lignin biosynthesis.

Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily "plastic" amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.

The multiple phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two distinct protein lineages.

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.

Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases.

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.

Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family.

Recently, a novel family of methyltransferases was identified in plants. Some members of this newly discovered and recently characterized methyltransferase family catalyze the formation of small-molecule methyl esters using S-adenosyl-L-Met (SAM) as a methyl donor and carboxylic acid-bearing substrates as methyl acceptors. These enzymes include SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase). Moreover, other members of this family of plant methyltransferases have been found to catalyze the N-methylation of caffeine precursors. The 3.0-A crystal structure of Clarkia breweri SAMT in complex with the substrate salicylic acid and the demethylated product S-adenosyl-L-homocysteine reveals a protein structure that possesses a helical active site capping domain and a unique dimerization interface. In addition, the chemical determinants responsible for the selection of salicylic acid demonstrate the structural basis for facile variations of substrate selectivity among functionally characterized plant carboxyl-directed and nitrogen-directed methyltransferases and a growing set of related proteins that have yet to be examined biochemically. Using the three-dimensional structure of SAMT as a guide, we examined the substrate specificity of SAMT by site-directed mutagenesis and activity assays against 12 carboxyl-containing small molecules. Moreover, the utility of structural information for the functional characterization of this large family of plant methyltransferases was demonstrated by the discovery of an Arabidopsis methyltransferase that is specific for the carboxyl-bearing phytohormone indole-3-acetic acid.

Evidence for positive selection on the floral scent gene isoeugenol-O-methyltransferase.

Isoeugenol-O-methyltransferase (IEMT) is an enzyme involved in the production of the floral volatile compounds methyl eugenol and methyl isoeugenol in Clarkia breweri (Onagraceae). IEMT likely evolved by gene duplication from caffeic acid-O-methyltransferase followed by amino acid divergence, leading to the acquisition of its novel function. To investigate the selective context under which IEMT evolved, maximum likelihood methods that estimate variable d(N)/d(S) ratios among lineages, among sites, and among a combination of both lineages and sites were utilized. Statistically significant support was obtained for a hypothesis of positive selection driving the evolution of IEMT since its origin. Subsequent Bayesian analyses identified several sites in IEMT that have experienced positive selection. Most of these positions are in the active site of IEMT and have been shown by site-directed mutagenesis to have large effects on substrate specificity. Although the selective agent is unknown, the adaptive evolution of this gene may have resulted in increased effectiveness of pollinator attraction or herbivore repellence.

Single mutations silence PGiC2 genes in two very recent allotetraploid species of Clarkia.

Our understanding of how polyploidy influences gene evolution is limited by the fact there have been few molecular descriptions of particular genes and their expression in polyploid plants and their diploid progenitors. Here we use evidence from sequencing of genomic DNA and cDNA obtained by reverse transcriptase-polymerase chain reaction and 3' rapid amplification of cDNA ends to describe PgiC genes and their expression in two allotetraploid species of the wildflower genus Clarkia, C. delicata and C. similis. PgiC encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9) and was duplicated in the ancestral stock of Clarkia, giving rise to paralogous genes PgiC1 and PgiC2. The active form of the PGIC enzyme is a dimer of like subunits. The electrophoretic patterns in the parent species show three bands of activity, representing two homodimers and a heterodimer of intermediate mobility, and are encoded by two genes. The electrophoretic patterns in the tetraploids also show three bands, but the tetraploids were expected to have multiple PGIC isozymes encoded by four genes. Our molecular studies demonstrated that each tetraploid has two PgiC1 and two PgiC2 genes, as predicted. One gene in each of them has been silenced by a single mutation, and a functional protein is no longer produced. In C. similis, PgiC2(mod) was silenced by a mutation of a single nucleotide in exon 5 that created a stop codon. In C. delicata, a polymorphism exists between a normal allele and a defective allele of PgiC2(epi) that has a deletion of a splice junction in intron 19 that results in the synthesis of a transcript lacking an entire exon, an example of exon skipping. The three-banded PGIC electrophoretic pattern of both tetraploid species arises because isozymes encoded by two or three of the genes comigrate. A very recent origin for both tetraploids is suggested by the near identity of several of their PgiC genes to their corresponding diploid orthologues and the absence of any acceleration in mutation rates. The problem of assessing genetic redundancy in tetraploids is discussed.

Structure and evolution of linalool synthase.

Plant terpene synthases constitute a group of evolutionarily related enzymes. Within this group, however, enzymes that employ two different catalytic mechanisms, and their associated unique domains, are known. We investigated the structure of the gene encoding linalool synthase (LIS), an enzyme that uses geranyl pyrophosphate as a substrate and catalyzes the formation of linalool, an acyclic monoterpene found in the floral scents of many plants. Although LIS employs one catalytic mechanism (exemplified by limonene synthase [LMS]), it has sequence motifs indicative of both LMS-type synthases and the terpene synthases employing a different mechanism (exemplified by copalyl diphosphate synthase [CPS]). Here, we report that LIS genes analyzed from several species encode proteins that have overall 40%-96% identity to each other and have 11 introns in identical positions. Only the region encoding roughly the last half of the LIS gene (exons 9-12) has a gene structure similar to that of the LMS-type genes. On the other hand, in the first part of the LIS gene (exons 1-8), LIS gene structure is essentially identical to that found in the first half of the gene encoding CPS. In addition, the level of similarity in the coding information of this region between the LIS and CPS genes is also significant, whereas the second half of the LIS protein is most similar to LMS-type synthases. Thus, LIS appears to be a composite gene which might have evolved from a recombination event between two different types of terpene synthases. The combined evolutionary mechanisms of duplication followed by divergence and/or "domain swapping" may explain the extraordinarily large diversity of proteins found in the plant terpene synthase family.