Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase-related proteins

Family GH13, also known as the alpha-amylase family, is the largest sequence-based family of glycoside hydrolases and groups together a number of different enzyme activities and substrate specificities acting on alpha-glycosidic bonds. This polyspecificity results in the fact that the simple membership of this family cannot be used for the prediction of gene function based on sequence alone. In order to establish robust groups that show an improved correlation between sequence and enzymatic specificity, we have performed a large-scale analysis of 1691 family GH13 sequences by combining clustering, similarity search and phylogenetic methods. About 80% of the sequences could be reliably classified into 35 subfamilies. Most subfamilies appear monofunctional (i.e. contain enzymes with the same substrate and the same product). The close examination of the other, apparently polyspecific, subfamilies revealed that they actually group together enzymes with strongly related (or even sometimes virtually identical) activities. Overall our subfamily assignment allows to set the limits for genomic function prediction on this large family of biologically and industrially important enzymes.     

A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants

Twelve neurologically normal infants (age 2.9+/-0.9 months) with peptic esophagitis (grade 2) who did not respond to cimetidine (in addition to positioning, cisapride, and Gaviscon) were treated with omeprazole, 0.5 mg/kg once a day, for 6 weeks. The effectiveness of omeprazole was evaluated in all infants by clinical assessment and endoscopy before and after treatment and by 24-hour gastric pH monitoring during treatment in seven infants. Omeprazole therapy led to a marked decrease in symptoms, endoscopic and histologic signs of esophagitis, and intragastric acidity.     

Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysproum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, has been determined by X-ray crystallography at a resolution of 2.7 A utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the presence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potential enzymatic cleavage is clearly distorted from the standard 4C1 chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L.O., Johnson, L.N., Machin, P. A., Phillips, D.C., & Tijan, T. (1974) J. Mol. Biol, 88, 349-371]. The distortion observed goes beyond the "sofa" conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.     

Detection of secondary structure elements in proteins by hydrophobic cluster analysis

Hydrophobic cluster analysis (HCA) is a protein sequence comparisonmethod based on -helical representations of the sequences wherethe size, shape and orientation of the clusters of hydrophobicresidues are primarily compared. The effectiveness of HCA hasbeen suggested to originate from its potential ability to focuson the residues forming the hydrophobic core of globular proteins.We have addressed the robustness of the bidimensional representationused for HCA in its ability to detect the regular secondarystructure elements of proteins. Various parameters have beenstudied such as those governing cluster size and limits, thehydrophobic residues constituting the clusters as well as thepotential shift of the cluster positions with respect to theposition of the regular secondary structure elements. The followingresults have been found to support the -helical bidimensionalrepresentation used in HCA: (i) there is a positive correlation(clearly above background noise) between the hydrophobic clustersand the regular secondary structure elements in proteins; (ii)the hydrophobic clusters are centred on the regular secondarystructure elements; (iii) the pitch of the helical representationwhich gives the best correspondence is that of an -helix. Thecorrespondence between hydrophobic clusters and regular secondarystructure elements suggests a way to implement variable gappenalties during the automatic alignment of protein sequences.     

Endogenous cellulases in animals: isolation of beta-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes

beta-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3' end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.     

The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, Mr = 50,513) is a unique 6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-alpha-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-alpha-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the Mr of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp41, Glu111, and Glu359, are required for GlvA activity. Asp41 is located at the C terminus of a betaalphabeta fold that may constitute the dinucleotide binding domain of the protein. Glu111 and Glu359 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-alpha-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-alpha-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.     

Comparison of three algorithms for the assignment of secondary structure in proteins: the advantages of a consensus assignment

Accurate assignments of secondary structures in proteins arecrucial for a useful comparison with theoretical predictions.Three major programs which automatically determine the locationof helices and strands are used for this purpose, namely DSSP,P-Curve and Define. Their results have been compared for a non-redundantdatabase of 154 proteins. On a residue per residue basis, thepercentage match score is only 63% between the three methods.While these methods agree on the overall number of residuesin each of the three states (helix, strand or coil), they differon the number of helices or strands, thus implying a wide discrepancyin the length of assigned structural elements. Moreover, thelength distribution of helices and strands points to the existenceof artefacts inherent to each assignment algorithm. To overcomethese difficulties a consensus assignment is proposed whereeach residue is assigned to the state determined by at leasttwo of the three methods. With this assignment the artefactsof each algorithm are attenuated. The residues assigned in thesame state by the three methods are better predicted than theothers. This assignment will thus be useful for analysing thesuccess rate of prediction methods more accurately.     

Domain evolution in the alpha-amylase family

Recently, we have characterized glial cultures derived from very early neurogenesis (E3) and found them to consist largely of early glioblastic or astroblastic cells with the capacity to differentiate into astrocytes given sufficient time in culture or with advancing age, i.e., cell passage. This study examines and compares the characteristics of astrocyte-enriched cultures derived from advanced embryonic ages (E15) in the chick embryonic cerebral hemispheres. We report several remarkable findings. 1) Mature astrocytes (GFAP+, vimentin-) appear as early as 5 days in vitro (DIV) in primary culture (P0). 2) Also apparent in primary cultures were extensive populations of neurons (neurofilament+; NF+) growing atop or in close proximity to mature astrocytes. 3) NF+ neurons disappeared after the first cell passage, and GFAP+ astrocytes were greatly diminished within two cell passages thereafter. 3) High concentrations of NGF were expressed, presumably by glial cells, in primary cultures through 14 DIV, declining to a low plateau through 27 DIV and remaining low, but measurable in subsequent cell passages. 4) At later cell passages (> 5) immature phenotypes of these same cell types continued to be expressed in E15CH cultures, i.e., positive staining for GFAP and vimentin and GFAP, GS, and NGF can all be detected on Western blots. We conclude from these findings that 1) mitotic multipotential neural cells are present within cerebral hemispheres even at late stages of development (E15); 2) neuroblasts and astroblasts have a reciprocal relationship requiring the presence of both cell types in order for mature expression of their phenotypes; 3) the NGF profile parallels the appearance and disappearance of neurons in E15 chick embryonic cerebral hemisphere primary cultures, strongly suggesting that this trophic factor may be involved in the mutually beneficial relationship between astrocytes and neurons.     

Structures and mechanisms of glycosyl hydrolases

The wealth of information provided by the recent structure determinations of many different glycosyl hydrolases shows that the substrate specificity and the mode of action of these enzymes are governed by exquisite details of their three-dimensional structures rather than by their global fold.     

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.