Lignin is, second to cellulose, the most abundant organic compound in the terrestrial biosphere. In different tree species, lignin content varies between 15 and 36% of the dry weight of wood (1). Lignin is a major constituent of cell walls of fibers and tracheary elements and provides these cells rigidity for structural support and impermeability for water transport. For the production of high-quality paper, lignin is considered as a negative factor because it must be extracted from the cellulose fraction by energy-requiring and polluting methods. For this reason, there is considerable interest in modifying lignin by genetic engineering to improve its extractability from wood (2-5).Lignin monomer biosynthesis starts with the deamination of phenylalanine to produce cinnamic acid (Fig. 1). Further enzymatic reactions include the hydroxylation of the aromatic ring, the methylation of selected phenolic hydroxyl groups, the activation of the cinnamic acids to cinnamoyl-CoA esters, and the reduction of these esters to cinnamaldehydes and cinnamyl alcohols. The precise order in which these reactions occur is not yet fully resolved. In dicotyledonous plants, lignin is composed mainly of guaiacyl (G) 1 and syringyl (S) units that are monomethoxylated (C-3) and dimethoxylated (C-3, C-5) and derived from coniferyl alcohol and sinapyl alcohol, respectively. The lignin monomers are transported to the cell wall and are subsequently polymerized, resulting in the deposition of a crosslinked polymer. Although most of the lignin biosynthesis enzymes have been characterized at the molecular level, their precise role in determining lignin amount and composition still needs to be clarified.Based on in vitro data, it has been generally accepted that the methylation reactions in lignin biosynthesis occur exclusively at the cinnamic acid level and that they are catalyzed by a bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) (1). However, the analysis of transgenic tobacco and poplar with suppressed COMT activity has shown that

We have characterized the primary structures of the predominant N-linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chromatographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N-linked core. In the bulk of the N-glycans, the u-1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The u-1,6 arm shows a remarkable heterogeneity: in addition to a-1,2-Man and a-1,6-Man, the presence of a terminal mannose a-1,6-phosphodiester was observed. This latter substituent has not been characterized before on mannosidase-processed N-glycan and its function and synthesis pathway are entirely unknown. The predominant Nglycans on cellobiohydrolase I can be represented as follows : GlcMan,GIcNAc,, GlcMan,GlcNAc,, Man,GlcNAc,, ManPGlcMan,GlcNAc,, GlcMan,GlcNAc, and Man,GlcNAc,.Keywords: cellobiohydrolase I ; fungus ; N-glycans ; NMR ; Trichoderma reesei.Trichoderma reesei is a filamentous fungus capable of secreting high amounts of cellulose-degrading enzymes (up to 40 g/l by manipulated strains). This remarkable secretory capacity led to the idea to use this organism as a candidate host for the production of heterologous proteins [I]. However, it is not clear whether T. reesei can be used for production of pharmaceutically important glycoproteins since post-translational modifications by the fungus, such as glycosylation, could be a major problem for application. Oligosaccharides lacking sialic acid as terminal sugar residues are easily recognized by lectins circulating in blood and present on cells of the reticulo-endothelial system. This recognition leads to fast clearance, which prevents the therapeutic proteins reaching their target organs [2, 31. Furthermore, it has been demonstrated that foreign glycosyl structures often elicit an antigenic response [4].Only few data concerning glycosylation in filamentous fungi are available. They indicate high-mannose-type glycans of limited sizes compared with these synthesized by yeast [S-121. The identification by Salovuori et al. 1121 of Man,GlcNAc, and Man,GlcNAc, as the major N-glycans on 7: reesei cellobiohydrolase I (CBH I) suggested a possible analogy between fungal and mammalian oligosaccharide structures. For identical primary structures, in vitro and eventually in vivo extension of oligosaccharides on proteins secreted from this fungus to the complex type should be possible.Correspondence to R. Contreras Enzyme. a-mannosidase (EC 3.2.1.24).During the eukaryotic secretion process, proteins receive in the endoplasmic reticulum a common precursor oligosaccharide, namely Glc,Man,GlcNAc,. This precursor is transferred by an oligosaccharyl transferase to asparagine residues in the consensus sequence Asn-Xaa-(Ser or Thr), with Xaa representing any amino acid except proline. A battery of glycosidases and gl...

In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD:GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction of a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre.

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