Crystal structure of α-1,4-glucan lyase, a unique glycoside hydrolase family member with a novel catalytic mechanism.

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (ß/α)8-barrel catalytic domain with B and B' subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp(553) and Asp(665) are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite -1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.

Disruption of the L-arabitol dehydrogenase encoding gene in Aspergillus tubingensis results in increased xylanase production.

Fungal xylanases are of major importance to many industrial sectors, such as food and feed, paper and pulp, and biofuels. Improving their production is therefore highly relevant. We determined the molecular basis of an improved xylanase-producing strain of Aspergillus tubingensis that was generated by UV mutagenesis in an industrial strain improvement program. Using enzyme assays, gene expression, sequencing of the ladA locus in the parent and mutant, and complementation of the mutation, we were able to show that improved xylanase production was mainly caused by a chromosomal translocation that occurred between a subtilisin-like protease pepD gene and the L-arabitol dehydrogenase encoding gene (ladA), which is part of the L-arabinose catabolic pathway. This genomic rearrangement resulted in disruption of both genes and, as a consequence, the inability of the mutant to use L-arabinose as a carbon source, while growth on D-xylose was unaffected. Complementation with constitutively expressed ladA confirmed that the xylanase overproducing phenotype was mainly caused by loss of ladA function, while a knockout of xlnR in the UV mutant demonstrated that improved xylanase production was mediated by XlnR. This study demonstrates the potential of metabolic manipulation for increased production of fungal enzymes.

Crystal structure of bifunctional aldos-2-ulose dehydratase/isomerase from Phanerochaete chrysosporium with the reaction intermediate ascopyrone M.

The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5-d-anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed ß-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn(2+) and Mg(2+) located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn(2)(+)-depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn(2+) in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 Å apart. The dehydration reaction most likely follows an elimination mechanism, where Zn(2+) acts as a Lewis acid polarizing the C2 keto group of 1,5-d-anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155.

Unique regulatory mechanism for D-galactose utilization in Aspergillus nidulans.

This study describes two novel regulators, GalX and GalR, that control d-galactose utilization in Aspergillus nidulans. This system is unique for A. nidulans since no GalR homologs were found in other ascomycetes. GalR shares significant sequence identity with the arabinanolytic and xylanolytic regulators AraR and XlnR, but GalX is more distantly related.

Regulation of pentose utilisation by AraR, but not XlnR, differs in Aspergillus nidulans and Aspergillus niger.

Filamentous fungi are important producers of plant polysaccharide degrading enzymes that are used in many industrial applications. These enzymes are produced by the fungus to liberate monomeric sugars that are used as carbon source. Two of the main components of plant polysaccharides are L-arabinose and D-xylose, which are metabolized through the pentose catabolic pathway (PCP) in these fungi. In Aspergillus niger, the regulation of pentose release from polysaccharides and the PCP involves the transcriptional activators AraR and XlnR, which are also present in other Aspergilli such as Aspergillus nidulans. The comparative analysis revealed that the regulation of the PCP by AraR differs in A. nidulans and A. niger, whereas the regulation of the PCP by XlnR was similar in both species. This was demonstrated by the growth differences on L-arabinose between disruptant strains for araR and xlnR in A. nidulans and A. niger. In addition, the expression profiles of genes encoding L-arabinose reductase (larA), L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA) differed in these strains. This data suggests evolutionary changes in these two species that affect pentose utilisation. This study also implies that manipulating regulatory systems to improve the production of polysaccharide degrading enzymes, may give different results in different industrial fungi.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

HACA, the transcriptional activator of the unfolded protein response (UPR) in Aspergillus niger, binds to partly palindromic UPR elements of the consensus sequence 5'-CAN(G/A)NTGT/GCCT-3'.

The promoters of UPR target genes contain an unfolded protein response element (UPRE), which confers the stress inducibility to the gene, via an interaction with the transcription activator HACA. In the promoters of the Aspergillus ER-stress responsive genes bipA, cypB, pdiA, prpA, tigA, and hacA, a consensus sequence was identified, which was located close to the transcription start site of the gene (<81 bp), and corresponds to the sequence CAN(G/A)NTGT/GCCT. The UPRE is a partly palindromic sequence around a dispensable spacer nucleotide, followed by four highly conserved bases. By an in vitro selection procedure, an optimal binding site for HACA was isolated. This sequence, ACACGTGTCCT, resembles the UPRE but lacks the spacer nucleotide. It has a much higher binding affinity than the identified UPREs, and in vivo it behaves as a more powerful cis-acting element.