UDP-l-rhamnose is required for the biosynthesis of cell wall rhamnogalacturonan-I, rhamnogalacturonan-II, and natural compounds in plants. It has been suggested that the RHM2/MUM4 gene is involved in conversion of UDP-d-glucose to UDP-l-rhamnose on the basis of its effect on rhamnogalacturonan-I-directed development in Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 and RHM3, can be found in the A. thaliana genome. Here we present direct evidence that all three RHM proteins have UDP-d-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-d-glucose 3,5-epimerase, and UDP-4-keto-l-rhamnose 4-keto-reductase activities in the cytoplasm when expressed in the yeast Saccharomyces cerevisiae. Functional domain analysis revealed that the N-terminal region of RHM2 (RHM2-N; amino acids 1–370) has the first activity and the C-terminal region of RHM2 (RHM2-C; amino acids 371–667) has the two following activities. This suggests that RHM2 converts UDP-d-glucose to UDP-l-rhamnose via an UDP-4-keto-6-deoxy-d-glucose intermediate. Site-directed mutagenesis of RHM2 revealed that mucilage defects in MUM4-1 and MUM4-2 mutant seeds of A. thaliana are caused by abolishment of RHM2 enzymatic activity in the mutant strains and furthermore, that the GXXGXX(G/A) and YXXXK motifs are important for enzymatic activity. Moreover, a kinetic analysis of purified His₆-tagged RHM2-N protein revealed 5.9-fold higher affinity of RHM2 for UDP-d-glucose than for dTDP-d-glucose, the preferred substrate for dTDP-d-glucose 4,6-dehydratase from bacteria. RHM2-N activity is strongly inhibited by UDP-l-rhamnose, UDP-d-xylose, and UDP but not by other sugar nucleotides, suggesting that RHM2 maintains cytoplasmic levels of UDP-d-glucose and UDP-l-rhamnose via feedback inhibition by UDP-l-rhamnose and UDP-d-xylose.

In plants, serine residues in extensin, a cell wall protein, are glycosylated with O-linked galactose. However, the enzyme that is involved in the galactosylation of serine had not yet been identified. To identify the peptidyl serine O-α-galactosyltransferase (SGT), we chose Chlamydomonas reinhardtii as a model. We established an assay system for SGT activity using C. reinhardtii and Arabidopsis thaliana cell extracts. SGT protein was partially purified from cell extracts of C. reinhardtii and analyzed by tandem mass spectrometry to determine its amino acid sequence. The sequence matched the open reading frame XP_001696927 in the C. reinhardtii proteome database, and a corresponding DNA fragment encoding 748 amino acids (BAL63043) was cloned from a C. reinhardtii cDNA library. The 748-amino acid protein (CrSGT1) was produced using a yeast expression system, and the SGT activity was examined. Hydroxylation of proline residues adjacent to a serine in acceptor peptides was required for SGT activity. Genes for proteins containing conserved domains were found in various plant genomes, including A. thaliana and Nicotiana tabacum. The AtSGT1 and NtSGT1 proteins also showed SGT activity when expressed in yeast. In addition, knock-out lines of AtSGT1 and knockdown lines of NtSGT1 showed no or reduced SGT activity. The SGT1 sequence, which contains a conserved DXD motif and a C-terminal membrane spanning region, is the first example of a glycosyltransferase with type I membrane protein topology, and it showed no homology with known glycosyltransferases, indicating that SGT1 belongs to a novel glycosyltransferase gene family existing only in the plant kingdom.

UDP‐d‐glucuronic acid and UDP‐d‐xylose are required for the biosynthesis of glycosaminoglycan in mammals and of cell wall polysaccharides in plants. Given the importance of these glycans to some organisms, the development of a system for production of UDP‐d‐glucuronic acid and UDP‐d‐xylose from a common precursor could prove useful for a number of applications. The budding yeast Saccharomyces cerevisiae lacks an endogenous ability to synthesize or consume UDP‐d‐glucuronic acid and UDP‐d‐xylose. However, yeast have a large cytoplasmic pool of UDP‐d‐glucose that could be used to synthesize cell wall β‐glucan, as a precursor of UDP‐d‐glucuronic acid and UDP‐d‐xylose. Thus, if a mechanism for converting the precursors into the end‐products can be identified, yeast may be harnessed as a system for production of glycans. Here we report a novel S. cerevisiae strain that coexpresses the Arabidopsis thaliana genes UGD1 and UXS3, which encode a UDP‐glucose dehydrogenase (AtUGD1) and a UDP‐glucuronic acid decarboxylase (AtUXS3), respectively, which are required for the conversion of UDP‐d‐glucose to UDP‐d‐xylose in plants. The recombinant yeast strain was capable of converting UDP‐d‐glucose to UDP‐d‐glucuronic acid, and UDP‐d‐glucuronic acid to UDP‐d‐xylose, in the cytoplasm, demonstrating the usefulness of this yeast system for the synthesis of glycans. Furthermore, we observed that overexpression of AtUGD1 caused a reduction in the UDP‐d‐glucose pool, whereas coexpression of AtUXS3 and AtUGD1 did not result in reduction of the UDP‐d‐glucose pool. Enzymatic analysis of the purified hexamer His‐AtUGD1 revealed that AtUGD1 activity is strongly inhibited by UDP‐d‐xylose, suggesting that AtUGD1 maintains intracellular levels of UDP‐d‐glucose in cooperation with AtUXS3 via the inhibition of AtUGD1 by UDP‐d‐xylose.

ProteinO-glycosylation is essential for protein modification and plays important roles in eukaryotic cells.O-Mannosylation of proteins occurs in the filamentous fungusAspergillus. The structure and function of thepmtAgene, encoding proteinO-d-mannosyltransferase, which is responsible for the initialO-mannosylation reaction inAspergillus nidulans, was characterized. Disruption of thepmtAgene resulted in the reduction ofin vitroproteinO-d-mannosyltransferase activity to 6 % of that of the wild-type strain and led to underglycosylation of an extracellular glucoamylase. ThepmtAdisruptant exhibited abnormal cell morphology and alteration in carbohydrate composition, particularly reduction in the skeletal polysaccharides in the cell wall. The results indicate that PmtA is required for the formation of a normal cell wall inA. nidulans.

Summary: The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)‐containing polysaccharides and glycoconjugates, including O‐glycans, N‐glycans, fungal‐type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse‐genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of β‐Galf‐specific antibody EB‐A2 to O‐glycosylated WscA protein and galactomannoproteins. The results of an in‐vitro Galf antigen synthase assay revealed that GfsA has β1,5‐ or β1,6‐galactofuranosyltransferase activity for O‐glycans in glycoproteins, uses UDP‐d‐Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β‐galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O‐glycans in the Golgi.