<i>Arabidopsis thaliana</i> glucuronosyltransferase in family GT14

Arabinogalactan proteins are abundant cell-surface proteoglycans in plants and are involved in many cellular processes including somatic embryogenesis, cell-cell interactions, and cell elongation. We reported a glucuronosyltransferase encoded by Arabidopsis AtGlcAT14A, which catalyzes an addition of glucuronic acid residues to β-1,3- and β-1,6-linked galactans of arabinogalactan (Knoch et al. 2013). The knockout mutant of this gene resulted in the enhanced growth rate of hypocotyls and roots of seedlings, suggesting an involvement of AtGlcAT14A in cell elongation. AtGlcAt14A belongs to the family GT14 in the Carbohydrate Active Enzyme database (CAZy; www.cazy.org), in which a total of 11 proteins, including AtGLCAT14A, are classified from Arabidopsis thaliana. In this paper, we report the enzyme activities for the rest of the Arabidopsis GT14 isoforms, analyzed in the same way as for AtGlcAT14A. Evidently, two other Arabidopsis GT14 isoforms, At5g15050 and At2g37585, also possess the glucuronosyltransferase activity adding glucuronic acid residues to β-1,3- and β-1,6-linked galactans. Therefore, we named At5g15050 and At2g37585 as AtGlcAT14B and AtGlcAT14C, respectively

Arabinogalactan proteins:focus on carbohydrate active enzymes

Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/) involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development

Arabinogalactan glycosyltransferases target to a unique subcellular compartment that may function in unconventional secretion in plants

We report that fluorescently tagged arabinogalactan glycosyltransferases target not only the Golgi apparatus but also uncharacterized smaller compartments when transiently expressed in Nicotiana benthamiana. Approximately 80% of AtGALT31A [Arabidopsis thaliana galactosyltransferase from family 31 (At1g32930)] was found in the small compartments, of which, 45 and 40% of AtGALT29A [Arabidopsis thaliana galactosyltransferase from family 29 (At1g08280)] and AtGlcAT14A [Arabidopsis thaliana glucuronosyltransferase from family 14 (At5g39990)] colocalized with AtGALT31A, respectively; in contrast, N-glycosylation enzymes rarely colocalized (3-18%), implicating a role of the small compartments in a part of arabinogalactan (O-glycan) biosynthesis rather than N-glycan processing. The dual localization of AtGALT31A was also observed for fluorescently tagged AtGALT31A stably expressed in an Arabidopsis atgalt31a mutant background. Further, site-directed mutagenesis of a phosphorylation site of AtGALT29A (Y144) increased the frequency of the protein being targeted to the AtGALT31A-localized small compartments, suggesting a role of Y144 in subcellular targeting. The AtGALT31A localized to the small compartments were colocalized with neither SYP61 (syntaxin of plants 61), a marker for trans-Golgi network (TGN), nor FM4-64-stained endosomes. However, 41% colocalized with EXO70E2 (Arabidopsis thaliana exocyst protein Exo70 homolog 2), a marker for exocyst-positive organelles, and least affected by Brefeldin A and Wortmannin. Taken together, AtGALT31A localized to small compartments that are distinct from the Golgi apparatus, the SYP61-localized TGN, FM4-64-stained endosomes and Wortmannin-vacuolated prevacuolar compartments, but may be part of an unconventional protein secretory pathway represented by EXO70E2 in plants

Recombinant production and characterization of six novel GH27 and GH36 alpha-galactosidases from Penicillium subrubescens and their synergism with a commercial mannanase during the hydrolysis of lignocellulosic biomass

alpha-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular alpha-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All alpha-galactosidases exhibited high affinity to pNP alpha Gal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining alpha-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other alpha-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal alpha-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses.Peer reviewe

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 β-d-xylosidase/α-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a β-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-β-d-xylopyranoside (pNPX), para-nitrophenyl-α-L-arabinofuranoside (pNPA), β-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2–4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I − V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40°C and at pH 4.0–8.0 and showed maximum activity at pH 5.5 and 50°C. K(m) and k(cat) for pNPX were 15.6 ± 4.2 mM and 60.6 ± 10.8 s(-1), respectively, and substrate inhibition became apparent above 18 mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for β-d-xylosidase (XynB3) from Geobacillus stearothermophilus T‒6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn(2+) and Ag(+), which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg(505)Tyr(506) present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.Peer reviewe

Vanillic acid and methoxyhydroquinone production from guaiacyl units and related aromatic compounds using Aspergillus niger cell factories

Background The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved. Results Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds. Conclusions This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories.Peer reviewe

Comparative characterization of nine novel GH51, GH54 and GH62 α-l-arabinofuranosidases from Penicillium subrubescens

α-l-Arabinofuranosidases (ABFs) are important enzymes in plant biomass degradation with a wide range of applications. The ascomycete fungus Penicillium subrubescens has more α-l-arabinofuranosidase-encoding genes in its genome compared to other Penicillia. We characterized nine ABFs from glycoside hydrolase (GH) families GH51, GH54 and GH62 from this fungus and demonstrated that they have highly diverse specificity and activity levels, indicating that the expansion was accompanied by diversification of the enzymes. Comparison of the substrate preference of the enzymes to the expression of the corresponding genes when the fungus was grown on either of two plant biomass substrates did not show a clear correlation, suggesting a more complex regulatory system governing l-arabinose release from plant biomass by P. subrubescens