NMR analysis of the binding mode of two fungal endo-ß-1,4-mannanases from GH5 and GH26 families.

The enzymatic digestion of the main components of lignocellulosic biomass, including plant cell wall mannans, constitutes a fundamental step in the renewable biofuel production, with great potential benefit in the industrial field. Despite several reports of X-ray structures of glycoside hydrolases, how polysaccharides are specifically recognized and accommodated in the enzymes binding site still remains a pivotal matter of research. Within this frame, NMR spectroscopic techniques provide key binding information, complementing and/or enhancing the structural view by X-ray crystallography. Here we provide deep insights into the binding mode of two endo-ß-1,4 mannanases from the coprophilous ascomycete Podospora anserina, PaMan26A and PaMan5A, involved in the hydrolysis of plant cell wall mannans and heteromannans. The investigation at a molecular level of the interaction between the wild-type enzymes and inactive mutants with manno-oligosaccharides, revealed a different mode of action among the two glycoside hydrolases most likely due to the presence of the additional and peculiar -4 subsite in the PaMan26A binding pocket.

Novel ß-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans.

Many filamentous fungi produce ß-mannan-degrading ß-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel ß-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free ß-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with ß-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-ß-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. The action of Man5C toward ß-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when ß-mannans were the sole carbon source, indicating that Man134A is involved in ß-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of ß-mannan degradation that is enhanced by the novel ß-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-ß-1,4-mannanases.

The class I α1,2-mannosidases of Caenorhabditis elegans.

During the biosynthesis of N-glycans in multicellular eukaryotes, glycans with the compositions Man(5)GlcNAc(2-3) are key intermediates. However, to reach this 'decision point', these N-glycans are first processed from Glc(3)Man(9)GlcNAc(2) through to Man(5)GlcNAc(2) by a number of glycosidases, whereby up to four α1-2-linked mannose residues are removed by class I mannosidases (glycohydrolase family 47). Whereas in the yeast Saccharomyces cerevisiae there are maximally three members of this protein family, in higher organisms there are multiple class I mannosidases residing in the endoplasmic reticulum and Golgi apparatus. The genome of the model nematode Caenorhabditis elegans encodes seven members of this protein family, whereby four are predicted to be classical processing mannosidases and three are related proteins with roles in quality control. In this study, cDNAs encoding the four predicted mannosidases were cloned and expressed in Pichia pastoris and the activity of these enzymes, designated MANS-1, MANS-2, MANS-3 and MANS-4, was verified. The first two can, dependent on the incubation time, remove three to four residues from Man(9)GlcNAc(2), whereas the action of the other two results in the appearance of the B isomer of Man(8)GlcNAc(2); together the complementary activities of these enzymes result in processing to Man(5)GlcNAc(2). With these data, another gap is closed in our understanding of the N-glycan biosynthesis pathway of the nematode worm.

Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A.

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-beta-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 angstroms resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of beta-stand beta4 and Glu231 at the C terminus of beta7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 interacts with the axial OH-C(2) in-2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.

Intact {alpha}-1,2-endomannosidase is a typical type II membrane protein.

Rat endomannosidase is a glycosidic enzyme that catalyzes the cleavage of di-, tri-, or tetrasaccharides (Glc(1-3)Man), from N-glycosylation intermediates with terminal glucose residues. To date it is the only characterized member of this class of endomannosidic enzymes. Although this protein has been demonstrated to localize to the Golgi lumenal membrane, the mechanism by which this occurs has not yet been determined. Using the rat endomannosidase sequence, we identified three homologs, one each in the human, mouse, and rat genomes. Alignment of the four encoded protein sequences demonstrated that the newly identified sequences are highly conserved but differed significantly at the N-terminus from the previously reported protein. In this study we have cloned two novel endomannosidase sequences from rat and human cDNA libraries, but were unable to amplify the open reading frame of the previously reported rat sequence. Analysis of the rat genome confirmed that the 59- and 39-termini of the previously reported sequence were in fact located on different chromosomes. This, in combination with our inability to amplify the previously reported sequence, indicated that the N-terminus of the rat endomannosidase sequence previously published was likely in error (a cloning artifact), and that the sequences reported in the current study encode the intact proteins. Furthermore, unlike the previous sequence, the three ORFs identified in this study encode proteins containing a single N-terminal transmembrane domain. Here we demonstrate that this region is responsible for Golgi localization and in doing so confirm that endomannosidase is a type II membrane protein, like the majority of other secretory pathway glycosylation enzymes.

Unique metal dependency of cytosolic alpha-mannosidase from Thermotoga maritima, a hyperthermophilic bacterium.

A putative cytosolic alpha-mannosidase gene from a hyperthermophilic marine bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The purified recombinant enzyme appeared to be a homodimer of a 110-kDa subunit. The enzyme showed metal-dependent ability to hydrolyze p-nitrophenyl-alpha-D-mannopyranoside. In the absence of a metal, the enzyme was inactive. Cobalt and cadmium supported high activity (60 U/mg at 70 degrees C), while the activity with zinc and chromium was poor. Cobalt (0.8 mol) bound to 1 mol monomer with a K(d) of 70 microM. The optimum pH and temperature were 6.0 and 80 degrees C, respectively. The activity was inhibited by swainsonine, but not by 1-deoxymannojirimycin, which is in agreement with the features of cytosolic alpha-mannosidase.

Molecular identification of family 38 alpha-mannosidase of Bacillus sp. strain GL1, responsible for complete depolymerization of xanthan.

When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, alpha-mannosidase exhibiting activity toward p-nitrophenyl-alpha-D-mannopyranoside (pNP-alpha-D-Man) was produced intracellularly. The 350-kDa alpha-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-alpha-D-Man (Km = 0.49 mM) and D-mannosyl-(alpha-1,3)-D-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for alpha-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of alpha-mannosidases belonging to glycoside hydrolase family 38.

Sequence and structural aspects of functional diversification in class I alpha-mannosidase evolution.

MOTIVATION: Class I alpha-mannosidases comprise a homologous and functionally diverse family of glycoside hydrolases. Phylogenetic analysis based on an amino acid sequence alignment of the catalytic domain of class I alpha-mannosidases reveals four well-supported phylogenetic groups within this family. These groups include a number of paralogous members generated by gene duplications that occurred as far back as the initial divergence of the crown-group of eukaryotes. Three of the four phylogenetic groups consist of enzymes that have group-specific biochemical specificity and/or sites of activity. An attempt has been made to uncover the role that natural selection played in the sequence and structural divergence between the phylogenetically and functionally distinct Endoplasmic Reticulum (ER) and Golgi apparatus groups. RESULTS: Comparison of site-specific amino acid variability profiles for the ER and Golgi groups revealed statistically significant evidence for functional diversification at the sequence level and indicated a number of residues that are most likely to have played a role in the functional divergence between the two groups. The majority of these sites appear to contain residues that have been fixed within one organelle-specific group by positive selection. Somewhat surprisingly these selected residues map to the periphery of the alpha-mannosidase catalytic domain tertiary structure. Changes in these peripherally located residues would not seem to have a gross effect on protein function. Thus diversifying selection between the two groups may have acted in a gradual manner consistent with the Darwinian model of natural selection. CONTACT: bishogr@millsaps.edu.

Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells.

The study of the glycosylation pathway of a mannosylphosphoryldolichol-deficient CHO mutant cell line (B3F7) reveals that truncated Glc(0-3)Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse-chase experiments indicate that these newly synthesized glycoproteins are retained in intracellular compartments and converted to Man4GlcNAc2 species. In this paper, we demonstrate that the alpha1,2 mannosidase, which is involved in the processing of Man5GlcNAc2 into Man4GlcNAc2, is located in the rough endoplasmic reticulum. The enzyme was shown to be inhibited by kifunensine and deoxymannojirimycin, indicating that it is a class I mannosidase. In addition, Man4GlcNAc2 species were produced at the expense of Glc1Man5GlcNAc2 species. Thus, the trimming of Man5GlcNAc2 to Man4GlcNAc2, which is catalyzed by this mannosidase, could be involved in the control of the glucose-dependent folding pathway.

Crystallization and preliminary X-ray crystallographic analysis of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5.

Crystals of the catalytic core domain of a Trichoderma reesei beta-mannanase belonging to glycoside hydrolase family 5 have been grown by the sitting-drop method at room temperature using ammonium sulfate as precipitant. The crystals grow as thin colourless plates and belong to space group P21, with unit-cell parameters a = 50.0, b = 54.3, c = 60.2 A, beta = 111.3 degrees, and have a single monomer of mannanase in the asymmetric unit. Native data to 2.0 A resolution have been collected at room temperature using synchrotron radiation. Data for a platinum derivative have been collected to 1.65 A at 110 K in a very short time at the CCLRC Daresbury synchrotron source, using a charge-coupled device (CCD) as detector.

Identification and analysis of a class 2 alpha-mannosidase from Aspergillus nidulans.

A Class 2 alpha-mannosidase gene was cloned and sequenced from the filamentous fungus Aspergillus nidulans. A portion of the gene was amplified using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat ER/cytosolic Class 2 protein sequences. The PCR amplification product was used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns. The predicted 1049 amino acid reading frame contained six potential N-glycosylation sites and encoded a protein of 118 kDa. The protein sequence did not appear to encode a typical fungal signal sequence or membrane spanning domain. Although the cellular location of the A.nidulans mannosidase was not determined, experimental evidence suggested that it was located within a subcellular organelle. The Matchbox sequence similarity matrix indicated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mannosidases (Rij = 0.43) than the rat and yeast sequences were to each other (Rij = 0.29). These three enzymes were found to be distantly related to other Class 2 sequences, and compose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.

Mammalian alpha-mannosidases--multiple forms but a common purpose?

Previously, alpha-mannosidases were classified as enzymes that process newly formed N-glycans or degrade mature glycoproteins. In this review, we suggest that two endoplasmic reticulum (ER) alpha-mannosidases, previously assigned processing roles, have important catabolic activities. Based on new evidence, we propose that the ER/cytosolic mannosidase is involved in the degradation of dolichol intermediates that are not needed for protein glycosylation, whereas the soluble form of Man9-mannosidase is responsible for the degradation of glycans on defective or malfolded proteins that are specifically retained and broken down in the ER. The degradation of oligosaccharides derived from dolichol intermediates by ER/cytosolic mannosidase now explains why cats and cattle with alpha-mannosidosis store and excrete some unexpected oligosaccharides containing only one GlcNAc residue. Similarly, the action of ER/cytosolic mannosidase, followed by the action of the recently described human lysosomal alpha(1 --> 6)-mannosidase, together explain why alpha-mannosidosis patients store and excrete large amounts of oligosaccharides that resemble biosynthetic intermediates, rather than partially degraded glycans. The relative contributions of the lysosomal and extra-lysosomal catabolic pathways can be derived by comparing the ratio of trisaccharide Man beta (1 --> 4)GlcNAc beta (1 --> 4)GlcNAc to disaccharide Man beta (1 --> 4)GlcNAc accumulated in tissues from goats with beta-mannosidosis. A similar determination in human beta-mannosidosis patients is not possible because the same intermediate, Man beta (1 --> 4)-GlcNAc is a product of both pathways. Based on inhibitor studies with pyranose and furanose analogues, alpha-mannosidases may be divided into two groups. Those in Class 1 are (1 --> 2)-specific enzymes like Golgi mannosidase I, whereas those in Class 2, like lysosomal alpha-mannosidase, can hydrolyse (1 --> 2), (1 --> 3) and (1 --> 6) linkages. A similar classification has recently been derived by others from protein sequence homologies. Based on this new classification of the alpha-mannosidases, it is possible to speculate about their probable evolution from two primordial genes. The first would have been a Class 1 ER enzyme involved in the degradation of glycans on incompletely assembled or malfolded glycoproteins. The second would have been a Class 2 lysosomal enzyme responsible for turnover. Later, other alpha-mannosidases, with new processing or catabolic functions, would have developed from these, by loss or gain of critical insertion or retention sequences, to yield the full complement of alpha-mannosidases known today.

alpha-D-mannosidase forms in chicken liver.

Two forms (I and II) of alpha-D-mannosidase have been separated by ion-exchange chromatography on DEAE-cellulose from embryonic chicken liver. A third form (III), which is absent in embryos, was also separated from 4-day-old chickens. The optimum pH of form I is at pH 5.0. Form II is named "neutral" because it shows maximal activity at pH 6.5. The optimum pH of form III is 4.5. Forms I and III are heat-stable at 50 degrees C for 1 hr, whereas form II is very unstable under these conditions. Zn2+ and Mg2+ have been found to increase the alpha-D-mannosidase activity of forms I and II. In contrast, Co2+ increases mannosidase I activity and inhibits form II from 18-day-old embryos. alpha-Methyl-D-mannoside, N-acetyl-D-mannosamine and D-mannosamine were found to be inhibitors of both forms I and II. "Neutral" mannosidase was also inhibited by chloride. Competitive inhibition by D-mannose was also studied and Ki values are given.