Molecular basis of hyper-thermostability in the thermophilic archaeal aldolase MfnB.

Methanogenic archaea are chemolithotrophic prokaryotes that can reduce carbon dioxide with hydrogen gas to form methane. These microorganisms make a significant contribution to the global carbon cycle, with methanogenic archaea from anoxic environments estimated to contribute > 500 million tons of global methane annually. Archaeal methanogenesis is dependent on the methanofurans; aminomethylfuran containing coenzymes that act as the primary C1 acceptor molecule during carbon dioxide fixation. Although the biosynthetic pathway to the methanofurans has been elucidated, structural adaptations which confer thermotolerance to Mfn enzymes from extremophilic archaea are yet to be investigated. Here we focus on the methanofuran biosynthetic enzyme MfnB, which catalyses the condensation of two molecules of glyceralde-3-phosphate to form 4­(hydroxymethyl)-2-furancarboxaldehyde-phosphate. In this study, MfnB enzymes from the hyperthermophile Methanocaldococcus jannaschii and the mesophile Methanococcus maripaludis have been recombinantly overexpressed and purified to homogeneity. Thermal unfolding studies, together with steady-state kinetic assays, demonstrate thermoadaptation in the M. jannaschii enzyme. Molecular dynamics simulations have been used to provide a structural explanation for the observed properties. These reveal a greater number of side chain interactions in the M. jannaschii enzyme, which may confer protection from heating effects by enforcing spatial residue constraints.

A Malassezia pseudoprotease dominates the secreted hydrolase landscape and is a potential allergen on skin.

Malassezia globosa is abundant and prevalent on sebaceous areas of the human skin. Genome annotation reveals that M. globosa possesses a repertoire of secreted hydrolytic enzymes relevant for lipid and protein metabolism. However, the functional significance of these enzymes is uncertain and presence of these genes in the genome does not always translate to expression at the cutaneous surface. In this study we utilized targeted RNA sequencing from samples isolated directly from the skin to quantify gene expression of M. globosa secreted proteases, lipases, phospholipases and sphingomyelinases. Our findings indicate that the expression of these enzymes is dynamically regulated by the environment in which the fungus resides, as different growth phases of the planktonic culture of M. globosa show distinct expression levels. Furthermore, we observed significant differences in the expression of these enzymes in culture compared to healthy sebaceous skin sites. By examining the in situ gene expression of M. globosa's secreted hydrolases, we identified a predicted aspartyl protease, MGL_3331, which is highly expressed on both healthy and disease-affected dermatological sites. However, molecular modeling and biochemical studies revealed that this protein has a non-canonical active site motif and lacks measurable proteolytic activity. This pseudoprotease MGL_3331 elicits a heightened IgE-reactivity in blood plasma isolated from patients with atopic dermatitis compared to healthy individuals and invokes a pro-inflammatory response in peripheral blood mononuclear cells. Overall, our study highlights the importance of studying fungal proteins expressed in physiologically relevant environments and underscores the notion that secreted inactive enzymes may have important functions in influencing host immunity.