Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level.

Archaea represents the third domain of life, displaying a closer relationship with eukaryotes than bacteria. These microorganisms are valuable model systems for molecular biology and biotechnology. In fact, nowadays, methanogens, halophiles, thermophilic euryarchaeota, and crenarchaeota are the four groups of archaea for which genetic systems have been well established, making them suitable as model systems and allowing for the increasing study of archaeal genes’ functions. Furthermore, thermophiles are used to explore several aspects of archaeal biology, such as stress responses, DNA replication and repair, transcription, translation and its regulation mechanisms, CRISPR systems, and carbon and energy metabolism. Extremophilic archaea also represent a valuable source of new biomolecules for biological and biotechnological applications, and there is growing interest in the development of engineered strains. In this review, we report on some of the most important aspects of the use of archaea as a model system for genetic evolution, the development of genetic tools, and their application for the elucidation of the basal molecular mechanisms in this domain of life. Furthermore, an overview on the discovery of new enzymes of biotechnological interest from archaea thriving in extreme environments is reported.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate‐active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β‐mannanase/β‐1,3‐glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD⁺‐dependent GH109 with a previously unreported β‐N‐acetylglucosaminide/β‐glucoside specificity.Databases: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).