Mutant barley (1-->3,1-->4)-beta-glucan endohydrolases with enhanced thermostability

The similar three-dimensional structures of barley (1-->3)-beta-glucan endohydrolases and (1-->3,1-->4)-beta-glucan endohydrolases indicate that the enzymes are closely related in evolutionary terms. However, the (1-->3)-beta-glucanases hydrolyze polysaccharides of the type found in fungal cell walls and are members of the pathogenesis-related PR2 group of proteins, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall metabolism. The (1-->3)-beta-glucanases have evolved to be significantly more stable than the (1-->3,1-->4)-beta-glucanases, probably as a consequence of the hostile environments imposed upon the plant by invading microorganisms. In attempts to define the molecular basis for the differences in stability, eight amino acid substitutions were introduced into a barley (1-->3,1-->4)-beta-glucanase using site-directed mutagenesis of a cDNA that encodes the enzyme. The amino acid substitutions chosen were based on structural comparisons of the barley (1-->3)- and (1-->3,1-->4)-beta-glucanases and of other higher plant (1-->3)-beta-glucanases. Three of the resulting mutant enzymes showed increased thermostability compared with the wild-type (1-->3,1-->4)-beta-glucanase. The largest increase in stability was observed when the histidine at position 300 was changed to a proline (mutant H300P), a mutation that was likely to decrease the entropy of the unfolded state of the enzyme. Furthermore, the three amino acid substitutions which increased the thermostability of barley (1-->3,1-->4)-beta-glucanase isoenzyme EII were all located in the COOH-terminal loop of the enzyme. Thus, this loop represents a particularly unstable region of the enzyme and could be involved in the initiation of unfolding of the (1-->3,1-->4)-beta-glucanase at elevated temperatures.     

Evolution and differential expression of the (1-->3)-beta-glucan endohydrolase-encoding gene family in barley, Hordeum vulgare.

The (1-->3)-beta-D-glucan glucanohydrolases [(1-->3)-GGH; EC 3.2.1.39] of barley (Hordeum vulgare L., cv Clipper) are encoded by a small gene family. Amino acid sequences deduced from cDNA and genomic clones for six members of the family exhibit overall positional identities ranging from 44% to 78%. Specific DNA and oligodeoxyribonucleotide (oligo) probes have been used to demonstrate that the (1-->3)-GGH-encoding genes are differentially transcribed in young roots, young leaves and the aleurone of germinated grain. The high degree of sequence homology, coupled with characteristic patterns of codon usage and insertion of a single intron at a highly conserved position in the signal peptide region, indicate that the genes have shared a common evolutionary history. Similar structural features in genes encoding barley (1-->3,1-->4)-beta-glucan 4-glucanohydrolases [(1-->3,1-->4)-GGH; EC 3.2.1.73] further indicate that the (1-->3)-GGHs and (1-->3,1-->4)-GGHs are derived from a single 'super' gene family, in which genes encoding enzymes with related yet quite distinct substrate specificities have evolved, with an associated specialization of function. The (1-->3,1-->4)-GGHs mediate in plant cell wall metabolism through their ability to hydrolyse the (1-->3,1-->4)-beta-glucans that are the major constituents in barley walls, while the (1-->3)-GGHs, which are unable to degrade the plant (1-->3,1-->4)-beta-glucans, can hydrolyse the (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls.     

A transglycosylating 1,3(4)-beta-glucanase from rhodothermus marinus NMR analysis of enzyme reactions.

The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.     

A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley

The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.     

The A- and B-chains of carboxypeptidase I from germinated barley originate from a single precursor polypeptide

Carboxypeptidase I from germinated barley (Hordeum vulgare) grain consists of two peptide chains linked by disulfides; the A- and B-chains contain 266 and 148 amino acid residues, respectively (Sorensen, S. B., Breddam, K., and Svendsen, I. (1986) Carlsberg Res. Commun. 51, 475-485). A cDNA library prepared from mRNA isolated from scutella of 2-day germinated barley has now been screened with a mixed oligonucleotide encoding a peptide fragment of the A-chain. Nucleotide sequence analysis of a 1443-nucleotide pair cDNA clone revealed that both chains of the enzyme are translated from a single mRNA. The coding region of the A-chain is located at the 5'-end of the cDNA and is separated from the B-chain coding region by a 165-nucleotide pair linking region. The B-chain coding region is followed by a stop codon, a 187-nucleotide pair 3'-untranslated sequence, and a short polyadenylic acid tail. The results indicate that the A- and B-chains of barley carboxypeptidase I arise by endoproteolytic excision of a 55-residue linker peptide from a single precursor polypeptide chain. The putative linker peptide is rich in proline, lysine, and arginine residues, has an apparent pI of 11.9, and appears to be excised by cleavage of peptide bonds on the COOH-terminal side of serine residues.     

Regulation of genes encoding β-d-glucan glucohydrolases in barley (Hordeum vulgare)

Expression patterns of barley β - d -glucan glucohydrolase genes were monitored using cDNAs encoding isoenzymes ExoI and ExoII. The cDNAs were isolated from 5-day-old seedling libraries. The enzymes are encoded by a small gene family, in which marked differences in codon usage are evident. The cDNAs can be used as specific probes for two subfamilies of β - d -glucan glucohydrolase genes. Genes of both subfamilies are transcribed in the scutellum of germinated grain, in elongating coleoptiles, and in young roots and leaves. Low levels of mRNA for the isoenzyme ExoI gene subfamily could be detected in aleurone layers of germinated grain. Most of the β - d -glucan glucohydrolase activity can be extracted from tissues with dilute aqueous buffers. Enzyme activity is highest in young leaves and elongating coleoptiles, but is not well-correlated with mRNA levels. The expression patterns are consistent with proposed roles for β -glucan glucohydrolases in the turnover or modification of cell-wall (1→3,1→4)- β - d -glucans in elongating coleoptiles and in young vegetative tissues.     

Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase.

BACKGROUND: Cell walls of the starchy endosperm and young vegetative tissues of barley (Hordeum vulgare) contain high levels of (1-->3,1-->4)-beta-D-glucans. The (1-->3,1-->4)-beta-D-glucans are hydrolysed during wall degradation in germinated grain and during wall loosening in elongating coleoptiles. These key processes of plant development are mediated by several polysaccharide endohydrolases and exohydrolases. RESULTS:. The three-dimensional structure of barley beta-D-glucan exohydrolase isoenzyme ExoI has been determined by X-ray crystallography. This is the first reported structure of a family 3 glycosyl hydrolase. The enzyme is a two-domain, globular protein of 605 amino acid residues and is N-glycosylated at three sites. The first 357 residues constitute an (alpha/beta)8 TIM-barrel domain. The second domain consists of residues 374-559 arranged in a six-stranded beta sandwich, which contains a beta sheet of five parallel beta strands and one antiparallel beta strand, with three alpha helices on either side of the sheet. A glucose moiety is observed in a pocket at the interface of the two domains, where Asp285 and Glu491 are believed to be involved in catalysis. CONCLUSIONS: The pocket at the interface of the two domains is probably the active site of the enzyme. Because amino acid residues that line this active-site pocket arise from both domains, activity could be regulated through the spatial disposition of the domains. Furthermore, there are sites on the second domain that may bind carbohydrate, as suggested by previously published kinetic data indicating that, in addition to the catalytic site, the enzyme has a second binding site specific for (1-->3, 1-->4)-beta-D-glucans.     

Differences in the thermostabilities of barley (1----3,1----4)-beta-glucanases are only partly determined by N-glycosylation.

Barley (1----3,1----4)-beta-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzyme EII carries 4% by weight carbohydrate and is more stable at elevated temperatures than isoenzyme EI, which has no associated carbohydrate. The relationship between carbohydrate content and thermostability has been investigated by treatment of the two isoenzymes with N-glycopeptidase F (EC 3.5.1.52). Removal of carbohydrate from isoenzyme EII results in a decrease in the enzyme's thermostability, but treatment of isoenzyme EI with the N-glycopeptidase F has no effect. In addition, removal of a single N-glycosylation site in isoenzyme EII (Asn190-Ala-Ser) by site-directed mutagenesis of the corresponding cDNA led to a reduction in thermostability, while the introduction of this site into isoenzyme EI enhanced stability. We conclude that N-glycosylation of Asn190 enhances the stability of isoenzyme EII at elevated temperatures, but that other factors related to their primary structures also contribute to the differences in thermostabilities of the barley (1----3,1----4)-beta-glucanases.     

Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate

Aureobasidium pullulans produced pullulan and melanin when grown in medium containing low nitrate levels. With high nitrate concentrations, however, this fungus produced a mixture of exopolysaccharides (EPS) without melanin synthesis. At 0.78 g l(-1) N as nitrate, where no melanin synthesis occurred, maximum EPS yields reached 6.92 g l(-1) and then decreased to the final yield of 2.36 g l(-1). Following melanin addition (0.1 g l(-1)), yields reached 7.02 g l(-1) at 48 h and fell to a final yield of 5.21 g l(-1). The EPS produced in high nitrate medium contained both pullulan and (1-->3)-beta-glucan, but only pullulan was produced with melanin-supplementation. With melanin addition a doubling of (1-->3)-beta-glucanase activity was observed in high nitrate medium compared to that without supplementation. On the other hand amylolytic activities disappeared in medium with melanin production or addition. Culture filtrates sustained a higher reducing capacity (RC) when melanin was present. Low RC appeared to reduce (1-->3)-beta-glucanase activity and increase amylolytic activities. Thus, higher RC appears to inhibit production/activity of amylose-degrading enzymes capable of degrading pullulan, and stimulates (1-->3)-beta-glucanase synthesis/activity, leading to a preferential accumulation of pullulan.     

Purification and chemical properties of two 1,3;1,4-beta-glucan endohydrolases from germinating barley

Two 1,3;1,4-beta-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3;1,4-beta-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants.