Johannes Freitag

TraH, a fungal decarboxylase from Penicillium crustosum , belongs to the isopenicillin N synthase (IPNS) subfamily of non-heme iron/α-ketoglutarate (αKG)-dependent enzymes. IPNS enzymes are characterized by N- and C-terminal insertions that can reshape the active site. However, the functional and mechanistic roles of these elements, particularly in fungal decarboxylases, remain largely unexplored. Here, we report crystal structures of TraH in complex with various substrates, revealing a N-terminal loop, serving as lid and undergoes substrate-dependent dynamic conformational rearrangements. Upon binding of crustosic acid, this lid loop forms a hydrogen-bonding network to stabilize a water molecule, which mediates interaction between the conserved K191 residue in the DSBH (double-stranded β-helix) core and the substrate. Mutagenesis and QM/MM metadynamics simulations suggest that proton transfer from K191 is mediated by water molecules stabilized by the lid loop, supporting a decarboxylation mechanism distinct from the canonical strategy of direct carboxylate stabilization by DBSH core-located basic residues in other αKG-dependent decarboxylases. Interestingly, this loop is dispensable for the desaturation of crustosic acid methyl ester, thereby pinpointing its essential role to the precise positioning of the carboxylate substrate for proton transfer during decarboxylation. Evolutionary and structural analyses reveal significant variation in lid loop composition across IPNS enzymes, indicating its contribution to substrate recognition and functional diversification. Overall, our findings uncover a regulatory element in iron/αKG-dependent enzymes and offer insights into how non-core structural elements contribute to catalytic mechanism and evolution.

Reversible lysine acetylation is a highly conserved post-translational modification across all domains of life controlling diverse cellular processes such as metabolism and gene expression. However, the regulation of protein acetylation remains poorly understood. Here, we report a regulatory system in Bacillus subtilis that controls the activity of the histone deacetylase (HDAC)-like protein AcuC, which has multiple substrates including acetyl-CoA synthetase and translation elongation factor. We show that AcuC is inhibited via formation of a stable complex with the hitherto uncharacterized protein AcuB. We furthermore demonstrate that the alarmone diadenosine tetraphosphate (Ap4A) binds to the cystathionine beta-synthase (CBS) domain of AcuB, thereby stabilizing AcuB and further enhancing the inhibition of AcuC. In summary, this study identifies AcuB as an Ap4A regulated deacetylation inhibitor, revealing a uncharacterized molecular mechanism to control HDAC-like proteins. Thus, the alarmone Ap4A modulates protein (de)acetylation, pointing towards a regulatory network that connects stress response, protein acetylation, and acetyl-CoA biosynthesis.

Mannosylerythritol lipids (MELs) are antibacterial surface-active molecules produced by several basidiomycetous fungi. Enzymes for MEL biosynthesis are encoded in a gene cluster composed of five genes, which are both necessary and sufficient for the biosynthesis of MELs. Here, we report that in two closely related biotrophic pathogens —Sporisorium reilianum f. sp. reilianum ( SrS ) and Sporisorium reilianum SRZ2 —MEL production is achieved through the combined activity of two gene clusters. While the larger cluster is required for production of the disaccharide moiety, for attachment of one acyl chain to the sugar backbone and for export of MELs, the smaller cluster encodes two enzymes for additional acylation of the sugar backbone and is essential to produce mature MELs in S. reilianum . The genes in the smaller cluster appear to be duplications of two of the original genes in the larger cluster, which are not expressed in SrS but in related fungi, e.g., Ustilago maydis . MELs made by SrS are tri-acylated and exhibit altered physical and chemical properties, making them interesting candidates for future biotechnological and pharmaceutical applications. In summary, our study showcases how duplication and diversification of MEL clusters can lead to the evolution of novel biosurfactants. IMPORTANCE: Secondary metabolites produced by fungi can act as weapons against competitors, can help access nutrients, or assist development and communication. One group of secondary metabolites are surface-active glycolipids that have a great potential as biodegradable detergents. Upon nitrogen starvation, biosynthesis of the glycolipid mannosylerythritol lipid (MEL) is induced by a gene cluster composed of five genes in the plant pathogen Ustilago maydis and related basidiomycetous fungi. This study shows that in the smut fungus Sporisorium reilianum f. sp. reilianum, the five genes of the MEL gene cluster were not sufficient to produce the isolated MEL variant. In contrast to conventional MELs, MELs of S. reilianum are tri-acylated at the mannose moiety with acyl groups in the range of C ₆ –C ₁₀ . These MELs exhibit altered physical and chemical properties, making them interesting novel candidates for future applications. Furthermore, we demonstrate that in S. reilianum, a combination of two gene clusters is necessary for MEL biosynthesis, which enables a glimpse into the evolutionary history of the altered MEL species.

Acetyl-CoA synthetase (Acs) generates acetyl-coenzyme A (Ac-CoA) but its excessive activity can deplete ATP and lead to a growth arrest. To prevent this, Acs is regulated through Ac-CoA-dependent feedback inhibition executed by Ac-CoA-dependent acetyltransferases such as AcuA in Bacillus subtilis. AcuA acetylates the catalytic lysine of AcsA turning the synthetase inactive. Here, we report that AcuA and AcsA form a tightly intertwined complex – the C-terminal domain binds to acetyltransferase domain of AcuA, while the C-terminus of AcuA occupies the CoA-binding site in the N-terminal domain of AcsA. Formation of the complex reduces AcsA activity in addition to the well-established acetylation of the catalytic lysine 549 in AcsA which we show can disrupt the complex. Thus, different modes of regulation accomplished through AcuA adjust AcsA activity to the concentrations of the different substrates of the reaction. In summary, our study provides detailed mechanistic insights into the regulatory framework underlying acetyl-CoA biosynthesis from acetate.

Itaconic acid belongs to the high-value precursors for the production of biomass-based industrial compounds. It originates from the tricarboxylic acid cycle, and depending on the organism, it is produced by different biosynthetic routes. The basidiomycete fungus Ustilago maydis synthesizes itaconic acid via isomerization of cis-aconitic acid to trans-aconitic acid, and subsequent decarboxylation catalyzed by the trans-aconitate decarboxylase Tad1, which belongs to the aspartase/fumarase superfamily. Since no other decarboxylase has been identified within this protein superfamily, Tad1 constitutes a novel type of decarboxylase. Here, we present high-resolution crystal structures of Tad1, which, together with mutational analysis and nuclear magnetic resonance spectroscopy measurements, provide insight into the molecular mechanism of Tad1-dependent decarboxylation. Specifically, our study shows that decarboxylation is favored in acidic conditions, requires protonation as well as migration of a double bond, and coincides with structural rearrangements in the catalytic center. In summary, our study elucidates the molecular mechanism underlying a novel type of enzymatic decarboxylation and provides a starting point for protein engineering aimed at optimizing the efficient production of itaconic acid.

Peroxisomes are single membrane-bounded oxidative organelles with various metabolic functions including β-oxidation of fatty acids. Peroxisomes of many species confine certain metabolic enzymes into sub-compartments sometimes visible as electron dense cores. Why these structures form is largely unknown. Here, we report that in the smut fungus Ustilago maydis detergent resistant core structures are enriched for different enzymes excluding several key enzymes of the β-oxidation pathway. This confinement contributes to generation of peroxisome subpopulations that differ in their enzyme content. We identify short amino acid motifs necessary and sufficient for protein self-assembly into aggregates in vitro. The motifs trigger enrichment in cores in vivo and are active in mammalian cells. Perturbation of core assembly via variation of such motifs affects peroxisome function in U. maydis strains challenged with fatty acids. Thus, protein core structures serve to compartmentalize the lumen of peroxisomes thereby preventing interference of biochemical reactions. Metabolic compartmentalization of peroxisomes via assembly of specific proteins may occur in other organisms as well.

The biotrophic fungus Ustilago maydis, which causes smut disease in maize, secretes numerous proteins upon plant colonization. Some of them, termed effectors, help to evade plant defenses and manipulate cellular processes within the host. The function of many proteins specifically secreted during infection remains elusive. In this study, we biochemically characterized one such protein, UMAG_00027, that is highly expressed during plant infection. We show that UMAG_00027 is a secreted protein with a lectin-like fold and therefore term it Llp1 (lectin-like-protein 1). Llp1 decorated the fungal cell wall of cells grown in axenic culture or proliferating in planta, which is in agreement with its potential sugar-binding ability. We were unable to identify the precise sugar moieties that are bound by Llp1. CRISPR/Cas9-mediated deletion of llp1 reveals that the gene is not essential for fungal virulence. A structural search shows the presence of several other lectin-like proteins in U. maydis that might compensate for the function of Llp1 in ∆llp1 mutants. We therefore speculate that Llp1 is part of a family of lectin-like proteins with redundant functions.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER–mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

Eukaryotic cells feature distinct membrane-enclosed organelles such as mitochondria and peroxisomes, each playing vital roles in cellular function and organization. These organelles are linked at membrane contact sites, facilitating interorganellar molecule and ion exchange. Most contact-forming proteins identified to date are membrane proteins or membrane-associated proteins, which can form very stable contacts. Recent findings suggest additional mechanistically distinct tethering events that arise from dual protein targeting. Proteins bearing targeting signals for multiple organelles, such as an N-terminal signal for mitochondria and a C-terminal signal for peroxisomes, function as tethers, fostering contacts by engaging targeting factors at both organelles. A number of dually targeted membrane proteins can contribute to contact site formation and transit from one organelle to the other as well. These interactions may enable the fine-tuning of organelle proximity, hence, adapting connections to meet varying physiological demands.