No abstract

SUMMARYA paradigm regarding rhamnogalacturonans II (RGII) is their strictly conserved structure within a given plant. We developed and employed a fast structural characterization method based on chromatography and mass spectrometry, allowing analysis of RGII side chains from microgram amounts of cell wall. We found that RGII structures are much more diverse than so far described. In chain A of wild-type plants, up to 45% of the L-fucose is substituted by L-galactose, a state that is seemingly uncorrelated with RGII dimerization capacity. This led us to completely reinvestigate RGII structures of the Arabidopsis thaliana fucose-deficient mutant mur1, which provided insights into RGII chain A biosynthesis, and suggested that chain A truncation, rather than L-fucose to L-galactose substitution, is responsible for the mur1 dwarf phenotype. Mass spectrometry data for chain A coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid, providing a plausible explanation for the puzzling RGII antibody recognition. The b-galacturonic acid of chain A exhibits up to two methyl etherifications in an organ-specific manner. Combined with variation in the length of side chain B, this gives rise to a family of RGII structures instead of the unique structure described up to now. These findings pave the way for studies on the physiological roles of modulation of RGII composition.

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe(2+) is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a K (m) of 9.1 microM and a k(cat) of 8.5 s(-1). A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.

Background: Human UDP-xylose synthase (hUXS1) is responsible for conversion of UDP-glucuronic acid to UDP-xylose.Results: Crystal structure, molecular dynamics simulations, and reaction course analysis give conclusive insight into the enzymatic mechanism in three catalytic steps.Conclusion: Distortion of sugar pyranose ring in bound substrate facilitates enzymatic reaction.Significance: A detailed mechanism for catalysis by hUXS1 is proposed.

The N-glycosylation of the model nematode Caenorhabiditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that ‘simple’ organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic sub-pool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a ‘bisecting’ β-galactose which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Not only do these residues constitute novel modifications of N-glycans, but these features may well be targets of nematoxic proteins produced as defence molecules by various fungal species.