Search

Selective adhesion of fungal cells to one another and to foreign surfaces is fundamental for the development of multicellular growth forms and the successful colonization of substrates and host organisms. Accordingly, fungi possess diverse cell wall-associated adhesins, mostly large glycoproteins, which present N-terminal adhesion domains at the cell surface for ligand recognition and binding. In order to function as robust adhesins, these glycoproteins must be covalently linkedto the cell wall via C-terminal glycosylphosphatidylinositol (GPI) anchors by transglycosylation. In this review, we summarize the current knowledge on the structural and functional diversity of so far characterized protein families of adhesion domains and set it into a broad context by an in-depth bioinformatics analysis using sequence similarity networks. In addition, we discuss possible mechanisms for the membrane-to-cell wall transfer of fungal adhesins by membrane-anchored Dfg5 transglycosidases.

(R)-Benzylsuccinate is the characteristic initial intermediate of anaerobic toluene metabolism, which is formed by a radical-type addition of toluene to fumarate. Its further degradation proceeds by activation to the coenzyme A (CoA)-thioester and β-oxidation involving a specific (R)-2-benzylsuccinyl-CoA dehydrogenase (BbsG) affiliated with the family of acyl-CoA dehydrogenases. In this report, we present the biochemical properties of electron transfer flavoproteins (ETFs) from the strictly anaerobic toluene-degrading species Geobacter metallireducens and Desulfobacula toluolica and the facultatively anaerobic bacterium Aromatoleum aromaticum. We determined the X-ray structure of the ETF paralogue involved in toluene metabolism of G. metallireducens, revealing strong overall similarities to previously characterized ETF variants but significantly different structural properties in the hinge regions mediating conformational changes. We also show that all strictly anaerobic toluene degraders utilize one of multiple genome-encoded related ETF paralogues, which constitute a distinct clade of similar sequences in the ETF family, for β-oxidation of benzylsuccinate. In contrast, facultatively anaerobic toluene degraders contain only one ETF species, which is utilized in all β-oxidation pathways. Our phylogenetic analysis of the known sequences of the ETF family suggests that at least 36 different clades can be differentiated, which are defined either by the taxonomic group of the respective host species (e.g., clade P for Proteobacteria) or by functional specialization (e.g., clade T for anaerobic toluene degradation). IMPORTANCE This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological electron acceptor for benzylsuccinyl-CoA dehydrogenase. While toluene-degrading denitrifying proteobacteria use a common ETF species, which is also used for other β-oxidation pathways, obligately anaerobic sulfate- or ferric-iron-reducing bacteria use specialized ETF paralogues for toluene degradation. Based on the structure and sequence conservation of these ETFs, they form a new clade that is only remotely related to the previously characterized members of the ETF family. An exhaustive analysis of the available sequences indicated that the protein family consists of several closely related clades of proven or potential electron-bifurcating ETF species and many deeply branching nonbifurcating clades, which either follow the host phylogeny or are affiliated according to functional criteria.

The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen., The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. At the cell wall level, enzyme activities are involved in postsynthesis polysaccharide modifications such as cleavage, elongation, branching, and cross-linking. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to participate in cell wall biosynthesis and specifically in polysaccharide remodeling. Among these proteins, the DFG family plays an essential role in controlling polar growth in yeast. In the filamentous fungus and opportunistic human pathogen Aspergillus fumigatus, the DFG gene family contains seven orthologous DFG genes among which only six are expressed under in vitro growth conditions. Deletions of single DFG genes revealed that DFG3 plays the most important morphogenetic role in this gene family. A sextuple-deletion mutant resulting from the deletion of all in vitro expressed DFG genes did not contain galactomannan in the cell wall and has severe growth defects. This study has shown that DFG members are absolutely necessary for the insertion of galactomannan into the cell wall of A. fumigatus and that the proper cell wall localization of the galactomannan is essential for correct fungal morphogenesis in A. fumigatus. IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen.

CYTH is a large protein superfamily that is conserved in all three domains of life with its unique triphosphate tunnel metalloenzyme (TTM) fold. Besides phosphatase functions, e.g. as RNA triphosphatase, inorganic polyphosphatase or thiamine triphosphatase, some CYTH orthologs cyclize nucleotide triphosphates to 3’,5’-cyclic nucleotides. So far, archaeal CYTH proteins are annotated as adenylyl cyclases although experimental evidence is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our initial in silico studies suggested a close relationship between archaeal CYTH enzymes and class IV adenylyl cyclases compared to the other CYTH-subclasses, but biochemical data showed no cyclic nucleotide production. Instead, our structural and functional analyses show a classical TTM behavior. The Ca2+-inhibited Michaelis complex indicates a two-metal ion reaction mechanism analogous to other TTMs. Different co-crystal structures of SaTTM further reveal conformational dynamics in SaTTM, let us to assume feedback inhibition in TTMs due to tunnel closure in the product state. Combining our structural insights with sequence-similarity network based in silico analysis, we further set out a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.Major highlights-CyaB-like class IV adenylyl cyclase homologs in archaea are triphosphatases.-The co-crystal structure of SaTTM in sulfate and triphosphate bound state revealed conformational transition of the TTM tunnel during catalysis.-Atomic insights into TTM inhibition by calcium and pyrophosphate.-In silico and structure-function analysis revealed the molecular determinant for functional diversification among CYTH proteins.

The nutritional alarmones ppGpp and pppGpp (collectively: (p)ppGpp) are nucleotide-based second messengers enabling bacteria to respond to environmental and stress conditions. Several bacterial species contain two highly homologous (p)ppGpp synthetases named RelP (SAS2, YwaC) and RelQ (SAS1, YjbM). It is established that RelQ forms homotetramers that are subject to positive allosteric regulation by pppGpp, but structural and mechanistic insights into RelP lack behind. Here we present a structural and mechanistic characterization of RelP. In stark contrast to RelQ, RelP is not allosterically regulated by pppGpp and displays a different enzyme kinetic behavior. This discrepancy is evoked by different conformational properties of the guanosine-substrate binding site (G-Loop) of both proteins. Our study shows how minor structural divergences between close homologues result in new functional features during the course of molecular evolution.