Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)₈-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ? α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.     

Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14–21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex]₁₂ and [Hex]₈, respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.     

Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi.

Endoglucanase Z from the phytopathogenic bacterium Erwinia chrysanthemi (strain 3937) was purified by affinity chromatography on microcrystalline cellulose Avicel PH101. A kinetic characterization using p-nitrophenyl beta-D-cellobioside and p-nitrophenyl beta-D-lactosde as substrates was conducted: endoglucanase Z exhibited Km values of 3 mM and 7.5 mM and Vm values of 129 and 40 nmol.min-1.mg-1 towards p-nitrophenyl beta-D-cellobioside (kcat = 0.1 s-1) and p-nitrophenyl beta-D-lactoside (kcat = 0.03 s-1), respectively). The hydrolysis of cellotetraitol by endoglucanase Z was followed by HPLC and 1H NMR. Results show that cellobiitol and beta-cellobiose are initially formed, demonstrating that the enzyme is acting by a molecular mechanism retaining the anomeric configuration. This suggests the involvement of a glycosyl-enzyme intermediate.     

Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a β-glucosidase/xylosidase enzyme

A -glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both -glucosidase and -xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.     

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

Background

Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels.

Methodology/Principal Findings

The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases ({"type":"entrez-protein","attrs":{"text":"ORF01222","term_id":"1178790230","term_text":"ORF01222"}}ORF01222, {"type":"entrez-protein","attrs":{"text":"ORF03896","term_id":"1178792974","term_text":"ORF03896"}}ORF03896, and {"type":"entrez-protein","attrs":{"text":"ORF01315","term_id":"1178790325","term_text":"ORF01315"}}ORF01315) exhibited the highest levels of up-regulation.

Conclusions/Significance

The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.     

Quantitative trait loci and comparative genomics of cereal cell wall composition

Quantitative trait loci (QTLs) affecting sugar composition of the cell walls of maize (Zea mays) pericarp were mapped as an approach to the identification of genes involved in cereal wall biosynthesis. Mapping was performed using the IBM (B73 x Mo17) recombinant inbred line population. There were statistically significant differences between B73 and Mo17 in content of xylose (Xyl), arabinose (Ara), galactose (Gal), and glucose. Thirteen QTLs were found, affecting the content of Xyl (two QTLs), Ara (two QTLs), Gal (five QTLs), Glc (two QTLs), Ara + Gal (one QTL), and Xyl + Glc (one QTL). The chromosomal regions corresponding to two of these, affecting Ara + Gal and Ara on maize chromosome 3, could be aligned with a syntenic region on rice (Oryza sativa) chromosome 1, which has been completely sequenced and annotated. The contiguous P1-derived artificial chromosome rice clones covering the QTLs were predicted to encode 117 and 125 proteins, respectively. Two of these genes encode putative glycosyltransferases, displaying similarity to carbohydrate-active enzyme database family GT4 (galactosyltransferases) or to family GT64 (C-terminal domain of animal heparan synthases). The results illustrate the potential of using natural variation, emerging genomic resources, and homeology within the Poaceae to identify candidate genes involved in the essential process of cell wall biosynthesis.