CreA mediates repression of the regulatory gene xlnR which controls the production of xylanolytic enzymes in Aspergillus nidulans.

The Aspergillus nidulans xlnR gene encodes a Zn(2)Cys(6) transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X(22), X(24) and X(34), and beta-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X(22)) and xlnB (encodes X(24)) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creA(d)30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.

A. niger protein "EstA", perhaps a new electrotactin, defines a new class of fungal esterases within the alpha/beta hydrolase fold superfamily.

Protein EstA from Aspergillus niger was characterized through a multifaced approach involving molecular biology, bioinformatics, biophysical, biochemical and enzymatical analyses. EstA was identified as the lead member, within the superfamily of proteins with an alpha/beta-hydrolase fold, of a new class of fungal esterases that also contains predicted homologs from other fungus species of known broad host-range pathogenicity.

Functional analysis of the transcriptional activator XlnR from Aspergillus niger.

The transcriptional activator XlnR from Aspergillus niger is a zinc binuclear cluster transcription factor that belongs to the GAL4 superfamily. Several putative structural domains in XlnR were predicted using database and protein sequence analysis. Thus far, only the functionality of the N-terminal DNA-binding domain has been determined experimentally. Deletion mutants of the xlnR gene were constructed to localize the functional regions of the protein. The results showed that a putative C-terminal coiled-coil region is involved in nuclear import of XlnR. After deletion of the C-terminus, including the coiled-coil region, XlnR was found in the cytoplasm, while deletion of the C-terminus downstream of the coiled-coil region resulted in nuclear import of XlnR. The latter mutant also showed increased xylanase activity, indicating the presence of a region with an inhibitory function in XlnR-controlled transcription. Previous findings had already shown that a mutation in the XlnR C-terminal region resulted in transcription of the structural genes under non-inducing conditions. A regulatory model of XlnR is presented in which the C-terminus responds to repressing signals, resulting in an inactive state of the protein.

Aspergillus niger protein EstA defines a new class of fungal esterases within the alpha/beta hydrolase fold superfamily of proteins.

From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipase-acetylcholinesterase chimera template. The 2.1 A resolution structure of EstA reveals a canonical Ser/Glu/His catalytic triad located in a small pocket at the bottom of a large solvent-accessible, bowl-shaped cavity. Potential substrates selected by manual docking procedures were assayed for EstA activity. Consistent with the pocket geometry, preference for hydrolysis of short acyl/propyl chain substrates was found. Identification of close homologs from the genome of other fungi, of which some are broad host-range pathogens, defines EstA as the first member of a novel class of fungal esterases within the superfamily. Hence the structure of EstA constitutes a lead template in the design of new antifungal agents directed toward its pathogenic homologs.

EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan.

A novel gene, eglC, encoding an endoglucanase, was cloned from Aspergillus niger. Transcription of eglC is regulated by XlnR, a transcriptional activator that controls the degradation of polysaccharides in plant cell walls. EglC is an 858-amino-acid protein and contains a conserved C-terminal cellulose-binding domain. EglC can be classified in glycoside hydrolase family 74. No homology to any of the endoglucanases from Trichoderma reesei was found. In the plant cell wall xyloglucan is closely linked to cellulose fibrils. We hypothesize that the EglC cellulose-binding domain anchors the enzyme to the cellulose chains while it is cleaving the xyloglucan backbone. By this action it may contribute to the degradation of the plant cell wall structure together with other enzymes, including hemicellulases and cellulases. EglC is most active towards xyloglucan and therefore is functionally different from the other two endoglucanases from A. niger, EglA and EglB, which exhibit the greatest activity towards beta-glucan. Although the mode of action of EglC is not known, this enzyme represents a new enzyme function involved in plant cell wall polysaccharide degradation by A. niger.