Molecular characterization of a cDNA encoding caffeoyl-coenzyme A 3-O-methyltransferase ofStellaria longipes

A cDNA clone encodingS-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyl-transferase (EC 2.1.1.104; CCoAOMT) fromStellana longipes Goldie (long-stalked chick-weed) was isolated and studied. Structural analysis of both the nucleotide sequence and the predicted amino acid sequence suggests that our cloned sequence encoded a CCoAOMT enzyme ofStellaria longipes, which shared overall structural similarity with other plant CCoAOMTs but exhibited certain distinct characteristics. Southern blot hybridization and cloning analyses indicating a small CCoAOMT gene family in theStellana longipes genome and the absence of introns in the coding region of the cDNA-corresponding gene. Sequence variations in the coding region were found among three genotypes from geographically isolated populations. Higher levels of CCoAOMT mRNA were detected in stems and leaves than in roots. The cDNA-encoded protein expressed inEschendia coli was shown to utilize caffeoyl-CoA, but not caffeic acid or 5-hydroxy ferulic acid, as its substrate.     

Fusion of Dioxygenase and Lignin-binding Domains in a Novel Secreted Enzyme from Cellulolytic Streptomyces sp. SirexAA-E

Streptomyces sp. SirexAA-E is a highly cellulolytic bacterium isolated from an insect/microbe symbiotic community. When grown on lignin-containing biomass, it secretes SACTE_2871, an aromatic ring dioxygenase domain fused to a family 5/12 carbohydrate-binding module (CBM 5/12). Here we present structural and catalytic studies of this novel fusion enzyme, thus providing insight into its function. The dioxygenase domain has the core β-sandwich fold typical of this enzyme family but lacks a dimerization domain observed in other intradiol dioxygenases. Consequently, the x-ray structure shows that the enzyme is monomeric and the Fe(III)-containing active site is exposed to solvent in a shallow depression on a planar surface. Purified SACTE_2871 catalyzes the O₂-dependent intradiol cleavage of catechyl compounds from lignin biosynthetic pathways, but not their methylated derivatives. Binding studies show that SACTE_2871 binds synthetic lignin polymers and chitin through the interactions of the CBM 5/12 domain, representing a new binding specificity for this fold-family. Based on its unique structural features and functional properties, we propose that SACTE_2871 contributes to the invasive nature of the insect/microbial community by destroying precursors needed by the plant for de novo lignin biosynthesis as part of its natural wounding response.     

Cation dependent O-methyltransferases from rice

Two lower molecular mass OMT genes (ROMT-15 and -17) were cloned from rice and expressed in Escherichia coli as glutathione S-transferase fusion proteins. ROMT-15 and -17 metabolized caffeoyl-CoA, flavones and flavonols containing two vicinal hydroxyl groups, although they exhibited different substrate specificities. The position of methylation in both luteolin and quercetin was determined to be the 3′ hydroxyl group and myricetin and tricetin were methylated not only at 3′ but also at 5′ hydroxyl groups. ROMT-15 and -17 are cation-dependent and mutation of the predicted metal binding sites resulted in the loss of the enzyme activity, indicating that the metal ion has a critical role in the enzymatic methylation.     

The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana

The Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a key enzyme in lignin biosynthesis in plants. In this study we cloned the full-length cDNA of the Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene from jute using homology clone (primers were designed according to the sequence of CCoAOMT gene of other plants), and a modified RACE technique, subsequently named “CcCCoAOMT1”. Bioinformatic analyses showed that the gene is a member of the CCoAOMT gene family. Real-time PCR analysis revealed that the CcCCoAOMT1 gene is constitutively expressed in all tissues, and the expression level was greatest in stem, followed by stem bark, roots and leaves. In order to understand this gene's function, we transformed it into Arabidopsis thaliana; integration (one insertion site) was confirmed following PCR and southern hybridization. The over-expression of CcCCoAOMT1 in these transgenic A.thaliana plants resulted in increased plant height and silique length relative to non-transgenic plants. Perhaps the most important finding was that the transgenic Arabidopsis plants contained more lignin (20.44–21.26%) than did control plants (17.56%), clearly suggesting an important role of CcCCoAOMT1 gene in lignin biosynthesis. These data are important for the success of efforts to reduce jute lignin content (thereby increasing fiber quality) via CcCCoAOMT1 gene inhibition.