Targeting plant UBX proteins: AI-enhanced lessons from distant cousins.

Across all eukaryotic kingdoms, ubiquitin regulatory X (UBX) domain-containing adaptor proteins control the segregase cell division control protein 48 (CDC48), and thereby also control cellular proteostasis and adaptation. The structures and biological roles of UBX proteins in animals and fungi have garnered considerable attention. However, their counterparts in plants remain markedly understudied. Since 2021, the artificial intelligence (AI)-based algorithm AlphaFold has provided predictions of protein structural features that can be highly accurate. Predictions of the proteomes of all major model organisms are now freely accessible to the entire research community through user-friendly web interfaces. We propose that the combination of cross-kingdom comparison with AF analysis produces a wealth of testable hypotheses to inspire and guide experimental research on plant UBX domain-containing (PUX) proteins.

Novel Enzymes From the Red Sea Brine Pools: Current State and Potential.

The Red Sea is a marine environment with unique chemical characteristics and physical topographies. Among the various habitats offered by the Red Sea, the deep-sea brine pools are the most extreme in terms of salinity, temperature and metal contents. Nonetheless, the brine pools host rich polyextremophilic bacterial and archaeal communities. These microbial communities are promising sources for various classes of enzymes adapted to harsh environments - extremozymes. Extremozymes are emerging as novel biocatalysts for biotechnological applications due to their ability to perform catalytic reactions under harsh biophysical conditions, such as those used in many industrial processes. In this review, we provide an overview of the extremozymes from different Red Sea brine pools and discuss the overall biotechnological potential of the Red Sea proteome.

Versatile control of the CDC48 segregase by the plant UBX-containing (PUX) proteins.

In plants, AAA-adenosine triphosphatase (ATPase) Cell Division Control Protein 48 (CDC48) uses the force generated through ATP hydrolysis to pull, extract, and unfold ubiquitylated or sumoylated proteins from the membrane, chromatin, or protein complexes. The resulting changes in protein or RNA content are an important means for plants to control protein homeostasis and thereby adapt to shifting environmental conditions. The activity and targeting of CDC48 are controlled by adaptor proteins, of which the plant ubiquitin regulatory X (UBX) domain-containing (PUX) proteins constitute the largest family. Emerging knowledge on the structure and function of PUX proteins highlights that these proteins are versatile factors for plant homeostasis and adaptation that might inspire biotechnological applications.

Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase.

Environments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.2% salt) by single-cell genome sequencing. The extensive analysis of the selected gene helps demonstrate the potential of this culture-independent method. The enzyme was expressed in the bioengineered haloarchaeon Halobacterium sp. NRC-1 and characterized by X-ray crystallography and mutagenesis. The 2.6 Å crystal structure of the protein shows a trimeric arrangement. Within the γ-CA, several possible structural determinants responsible for the enzyme's salt stability could be highlighted. Moreover, the amino acid composition on the protein surface and the intra- and intermolecular interactions within the protein differ significantly from those of its close homologs. To gain further insights into the catalytic residues of the γ-CA enzyme, we created a library of variants around the active site residues and successfully improved the enzyme activity by 17-fold. As several γ-CAs have been reported without measurable activity, this provides further clues as to critical residues. Our study reveals insights into the halophilic γ-CA activity and its unique adaptations. The study of the polyextremophilic carbonic anhydrase provides a basis for outlining insights into strategies for salt adaptation, yielding enzymes with industrially valuable properties, and the underlying mechanisms of protein evolution.