ABSTRACT
The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed ß-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.
Subject(s)
Alcohol Dehydrogenase/chemistry , Bacteria/enzymology , Bacterial Proteins/chemistry , Crystallography, X-Ray/methods , Alcohol Dehydrogenase/metabolism , Asparagine/metabolism , Bacteria/chemistry , Bacterial Proteins/metabolism , Catalytic Domain , Models, Molecular , Polyethylene Glycols/metabolism , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Galactose/metabolism , Gene Expression Regulation, Fungal , Metabolic Networks and Pathways/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Models, Biological , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Aspergillus nidulans/metabolism , Aspergillus niger/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Pentoses/metabolism , Trans-Activators/metabolism , Amino Acid Sequence , Aspergillus nidulans/enzymology , Aspergillus nidulans/genetics , Aspergillus niger/enzymology , Aspergillus niger/genetics , Fungal Proteins/genetics , Industrial Microbiology , Molecular Sequence Data , Sequence Alignment , Trans-Activators/chemistry , Trans-Activators/geneticsABSTRACT
Activation of the unfolded protein response (UPR) in eukaryotes involves the splicing of an unconventional intron from the mRNA encoding the transcriptional activator of the pathway. In Saccharomyces cerevisiae a 252-nucleotide (nt) unconventional intron is spliced out of the transcript of HAC1, changing the 3' end of the HAC1 open reading frame and relieving the transcript from a translational block in a single step. The translational block is caused by the base pairing of part of the unconventional intron with the 5'-untranslated region (5'UTR). In Aspergillus niger and other aspergilli, the unconventional intron in hacA mRNA is only 20 nt long. Since this intron is part of a stable stem-loop structure, base pairing with the 5'UTR, in contrast to the case with yeast HAC1, is not possible. However, analysis of the hacA mRNA revealed a GC-rich inverted repeat (18 base pairings). Upon the activation of the UPR, the 5'UTR of hacA mRNA is truncated by 230 nt, removing the left part of this inverted repeat. This implies a similar release of a translational block as in the case of S. cerevisiae HAC1 but in two steps. The mechanism behind the 5' truncation, which does not take place in either yeast HAC1 or mammalian xbp1 mRNA, has been hitherto unknown. Here we show that during secretion stress in A. niger, hacA transcription starts from a new start site closer to the ATG, relieving the transcript from translational attenuation. This transcriptional switch is mediated by HacA itself and the unfolded protein response element 2 (UPRE2) in the hacA promoter.
Subject(s)
Aspergillus niger/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Endoplasmic Reticulum/metabolism , Fungal Proteins/metabolism , Protein Biosynthesis , Transcription, Genetic , 5' Untranslated Regions , Aspergillus niger/chemistry , Aspergillus niger/genetics , Base Sequence , Basic-Leucine Zipper Transcription Factors/chemistry , Basic-Leucine Zipper Transcription Factors/genetics , Binding Sites , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Response ElementsABSTRACT
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.
