Biology subjects

Daumann, L. J.

Publications and source records attributed to Daumann, L. J..

6 recordsLinked to original sources

bioRxiv2026

There is no convincing evidence that Methylobacterium extorquens AM1 can produce N-deoxyschizokinen A

It was recently reported that Methylobacterium extorquens AM1 produces the citrate- hydroxamate siderophore N-deoxyschizokinen A, identified by LC-HRMS. Multiple properties were inconsistent with the assignment: the feature eluted far later than the other schizokinen derivatives (17 min versus 6-8 min), a reversed-phase shift larger than a single-hydroxyl difference in a molecule can explain, further its accurate mass deviated from the calculated one by 28 ppm, well outside the error on the co-analyzed standards and its diagnostic m/z 105 and 77 fragments suggest a molecule with an aromatic moiety. A replicate comparison of identical samples in plastic versus glass autosampler vials was decisive: the m/z 387 feature was reproducibly present with plastic vials and absent with glass. We therefore conclude that the reported detection of N-deoxyschizokinen A in M. extorquens AM1 is an artifact, and recommend glass-vial and solvent-blank controls, an explicit accurate-mass threshold, and narrow MS/MS isolation when assigning trace siderophore-like features from complex extracts.

microbiology↗

bioRxiv2026

An Unprecedented Cluster in a Methanotroph Acetol Dehydrogenase

Biocatalytic metal-containing clusters are nanometer-sized chemical reactors that enable enzymes to perform chemistry far beyond what would be possible with amino acids alone 1,2. These clusters display a remarkable variability3 and elucidating their exact mechanisms is often difficult to determine, requiring input from a multitude of techniques ranging from spectroscopic to structural and theoretical methods. Thus, for unknown clusters, it is essential to accumulate, collate, and interpret as much information as possible from every relevant technique available. Here we report the discovery and in-depth, interdisciplinary characterization of an entirely novel, [Cu-4Fe-4S] cluster in the protein acetol dehydrogenase (AceDH). AceDH, isolated directly from Methylacidiphilum fumariolicum SolV cells, catalyzed the oxidation of acetol to methylglyoxal, proving its role in the 2-propanol/acetone metabolism of methanotrophs4-6. Structural, spectroscopic and electrochemical analyses reveal the [Cu-4Fe-4S] cluster has a unique three-dimensional- and electronic structure involving electronic coupling between the copper and one of the iron atoms, likely contributing to its high, +275 mV, redox potential. The binding site for the novel cluster is composed of two protein subunits and involves a novel motif. These findings expand the known repertoire of biological metal cofactors and provide insight into how heterometallic clusters are adapted for biological processes.

biochemistry↗

bioRxiv2025

ALKBH1 activity in vitro and human cell lines by isotope dilution mass spectrometry

AlkB homolog 1 (ALKBH1) is a member of the AlkB family of Fe(II) and -ketoglutarate (-KG)-dependent dioxygenases, known for its enzymatic activity on various nucleic acid substrates. Its known targets include N1-methyladenosine (m1A), N6-methyladenosine (m6A), N3-methylcytidine (m3C), and 5-methylcytosine (m5C) in RNA, as well as N6-methyladenine (N6-mA, 6mA) in DNA and the histone protein H2A. Moreover, dysregulation or dysfunction of ALKBH1 has been implicated in a broad spectrum of human diseases. In order to shed further light on the substrate scope and role of ALKBH1, we used quantitative mass spectrometry to assess its activity in vitro and in human cell lines. To study ALKBH1 activity on defined substrates in vitro, we enzymatically generated tRNAs specifically carrying the m3C32 (tRNA-ThrUGU and tRNA-SerUCN) and i6A37 (tRNA-SerUCN) modifications. Here we show that ALKBH1 reduces m3C, m1A and m5C in vitro in total extracts of tRNAs, but does not impact on rRNA modifications in human cell lines. However, upon overexpression or siRNA-mediated knock-down of alkbh1 in human cell lines no impact on the modification of total tRNA extracts or specifically enriched RNAs could be observed. In addition, varying the glucose and fetal bovine serum (FBS) concentration in the growth medium of HEK293T cells, in combination with alkbh1 siRNA-mediated knock-down, also shows no impact on the tRNA modification spectra. Based on our data, we conclude that in human cells lines grown under optimal conditions ALKBH1 does not play an important role in the demethylation of tRNAs.

biochemistry↗

bioRxiv2024

Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability

The DNA-dependent protease SPRTN maintains genome stability by degrading toxic DNA-protein crosslinks (DPCs). To understand how SPRTNs promiscuous protease activity is confined to the cleavage of crosslinked proteins, we reconstitute the repair of DPCs including their modification with SUMO and ubiquitin chains, using recombinant human proteins. We discover that DPC ubiquitylation strongly activates SPRTN independently of SPRTNs known ubiquitin-binding domains. Using protein structure prediction, MD simulations and NMR spectroscopy we reveal that ubiquitin binds to an interface at the back of SPRTNs protease domain, promoting an active conformation. Replacing key interfacial residues prevents ubiquitin-dependent activation of SPRTN, which leads to genomic instability and cell cycle defects in cells expressing hypomorphic SPRTN variants that cause premature aging and liver cancer in Ruijs-Aalfs syndrome patients. Collectively, our results demonstrate that SPRTN activation is coupled to the modification of the crosslinked protein, explaining how specificity is achieved during DPC repair.

biochemistry↗

bioRxiv2023

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs from methanotrophs and methylotrophs are able to incorporate different lanthanides (Lns) in their active site. The artificial dye-coupled assay showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Although this assay is widely used, there are limitations. It is not unusual that a pH of 9 is required and that activators like ammonium have to be added to the assay mixture which do not reflect the conditions inside the cell. Moreover, different Ln-MDH variants are not obtained by the direct isolation from the cells grown with the respective Ln, but by metal titration of the Ln in a partial-apo-MDH or by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDH and the assessment of the enzyme activity using the artificial dye-coupled assay. We compare these results with an adapted protein-coupled assay using the physiological partner and electron acceptor cytochrome cGJ (cyt cGJ) instead of artificial dyes. We demonstrate that, depending on the assay, two distinct trends are observed among the Ln series. The specific activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds the specific enzyme activity measured by the dye-coupled assay. This suggests that the early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

biochemistry↗

bioRxiv2022

Identification of a biosynthetic gene cluster encoding a novel lanthanide chelator in Methylorubrum extorquens AM1

Many bacteria secrete metallophores, low-molecular weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment.1 Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper.1 No lanthanide-specific metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla.2 Here, we report the biosynthetic machinery, the structure, and the physiological relevance of the first known lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. Beyond Ln, we anticipate our study to be a starting point for understanding how organisms acquire other f-block metals, the actinides.3 More broadly, the discovery of a lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for new functions, how metallophores acquire their metal specificity, and the complex relationship between metal homeostasis and fitness.

microbiology↗