Biology subjects

Groll, M.

Publications and source records attributed to Groll, M..

7 recordsLinked to original sources

bioRxiv2025

Identification and biosynthesis of xildivaline, a novel and widespread peptide deformylase inhibitor from Gammaproteobacteria

Xenorhabdus strains, Gram-negative bacteria pathogenic to insects and symbionts to nematodes of the genus Steinernema are prolific producers of various natural products. Here we describe the xisABCDE biosynthesis gene cluster from Xenorhabdus hominickii responsible for the production of xildivalines. These non-ribosomal peptide and polyketide hybrids act as peptide deformylase inhibitor (PDI) and occur also in other Gammaproteobacteria, especially Vibrio. Their structure and biosynthesis were fully elucidated despite their instability, highlighting a rare trans-methylation of their N-terminus. Subsequently, the structure of the responsible methyltransferase XisE and the peptide deformylase XisD, serving as resistance mechanism, were elucidated by X-ray crystallography, allowing insights into the function and the mode of action of this novel class of PDIs.

microbiology↗

bioRxiv2024

Identification, structure and function of the methyltransferase involved in the biosynthesis of the dithiolopyrrolone antibiotic xenorhabdin

Xenorhabdins (XRDs) are produced by Xenorhabdus species and are members of the dithiopyrrolone (DTP) class of natural products that have potent antibacterial, antifungal and anticancer activity. The amide moiety of their DTP core can be methylated or not to fine-tune the bioactivity properties. However, the enzyme responsible for the amide N-methylation remained elusive. Here, we identified and characterized the amide methyltransferase XrdM that is encoded nearly 600 kb away from the XRD gene cluster using proteomic analysis, methyltransferase candidate screening, gene deletion, and allied approaches. In addition, crystallographic analysis and site-directed mutagenesis proved that XrdM is completely distinct from the recently reported DTP methyltransferase DtpM, and that both have been tailored in a species-specific manner for DTP biosynthesis in Gram-negative/positive organisms. Our study expands the limited knowledge of post-NRPS amide methylation in DTP biosynthesis and reveals the evolution of two structurally completely different enzymes for the same reaction in different organisms.

microbiology↗

bioRxiv2022

Evolutionary Inspired Engineering of Megasynthetases

Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

synthetic biology↗

bioRxiv2022

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes produce a myriad of natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the biosynthetic gene clusters (BGCs) has limited the understanding of how natural products justify the bacterial niche specificity. Here we combine pangenome and sequence similarity networks to analyze BGCs from 45 XP species. The identified 1,000 BGCs belong to 176 families, over half of which are unknown. Eleven BGCs represent the most conserved families. We then homologously express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, insect virulence factors, or insect immune suppressors. These findings account for the functional basis of bacterial natural products in this tripartite relationship.

biochemistry↗

bioRxiv2021

Cooperative DNA base and shape recognition by the CCAAT-binding complex and its bZIP transcription factor HapX

The heterotrimeric CCAAT-binding complex (CBC) is a master regulator of transcription. It specifically recognizes the CCAAT-box, a fundamental eukaryotic promoter element. Certain fungi, like Aspergilli, encode a fourth CBC-subunit, HapX, to fine-tune expression of genes involved in iron metabolism. Although being a basic region leucine zipper with its own DNA recognition motif, HapX function strictly relies on the CBC. We here report two crystal structures of the CBC-HapX complex bound to DNA duplexes with distinct sequence and position of HapX sites. In either structure, a HapX dimer targets the nucleic acid downstream of the CCAAT-box and the leash-like N-terminus of the distal HapX subunit interacts with CBC and DNA. In vitro and in vivo analyses of HapX mutants support the structures, highlight the complex as an exceptional major and minor groove DNA binder, and enrich our understanding of the functional as well as structural plasticity of related complexes across species.

biochemistry↗

bioRxiv2019

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

biochemistry↗

bioRxiv2017

Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

biochemistry↗