Biology subjects

Vermeir, F. J.

Publications and source records attributed to Vermeir, F. J..

4 recordsLinked to original sources

bioRxiv2026

The molecular mechanism and activity of Kuenenia stuttgartiensis hydrazine synthase

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite into dinitrogen gas via the intermediates nitric oxide and hydrazine. To produce hydrazine, anammox bacteria harbor a biochemically unique enzyme: hydrazine synthase. Based on the hydrazine synthase crystal structure it was hypothesized that hydrazine is produced in a two-step mechanism. In this hypothesis, nitric oxide is first reduced to hydroxylamine (first half-reaction), followed by condensation of hydroxylamine with ammonium to hydrazine (second half-reaction). Here, we experimentally investigated the proposed molecular mechanism of hydrazine synthase and characterized and optimized the in vitro activity. First, we optimized the activity of isolated hydrazine synthase from anammox bacterium Kuenenia stuttgartiensis strain MBR1 via an anaerobic isolation method. We further compared hydrazine synthase activity measured via a coupled assay versus that of a newly established direct LC-MS assay. Next, the hypothesized second half-reaction was investigated via the direct LC-MS assay, quantifying biologically produced hydrazine from hydroxylamine and ammonium. Despite variation in hydrazine synthase activity across assays, we determined ammonium and hydroxylamine affinity and investigated product inhibition. Finally, we found that hydrazine synthesis from ammonium and hydroxylamine by isolated hydrazine synthase is oxygen-tolerant, strongly suggesting that the second half-reaction is initiated on an oxidized heme within hydrazine synthase. Taken together, the results corroborate that condensation of ammonium with hydroxylamine to form hydrazine is the second half-reaction of the proposed two-step mechanism for hydrazine synthesis by hydrazine synthase.

microbiology↗

bioRxiv2026

Quantification of hydrazine in biochemical assays and anammox bacteria using LC-MS

Hydrazine is an industrially valuable product. Strikingly, hydrazine is also a key intermediate in the energy metabolism of anaerobic ammonium-oxidizing (anammox) bacteria, where it is formed by hydrazine synthase. To study the molecular mechanism and activity of isolated hydrazine synthase, a sensitive, relatively fast and easy method to quantify hydrazine is needed. However, reported methods such as colorimetric assays, MALDI-TOF MS, and enzymatic conversion of hydrazine to dinitrogen gas, are either insensitive or laborious. In this study, we describe the validation and application of a fast and simple liquid chromatography-mass spectrometry (LC-MS) method to reproducibly quantify hydrazine produced by anammox hydrazine synthase. Hydrazine was derivatized with benzaldehyde, and directly injected onto a C18 column coupled to a Q-TOF MS. To increase assay performance, 15N2-hydrazine was included as internal standard. The response ratio of hydrazine was linearly proportional to the hydrazine concentration from 0.05-1 {micro}M with an average correlation coefficient of 0.9925. Intra- and inter-day accuracy lay between 88-113% and 95-105%, respectively. Intra- and inter-day precision (RSD, %) [≤] 11%. Hydrazine and derivatized hydrazine were stable when stored at -70{degrees}C or in the autosampler. We successfully applied the LC-MS method to determine hydrazine production by isolated hydrazine synthase and within cell lysate of anammox bacteria.

biochemistry↗

bioRxiv2025

Nitric oxide-forming nitrite reductases in the anaerobic ammonium oxidizer Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing (anammox) bacteria contribute to the global nitrogen cycle by removing fixed nitrogen from the environment. They do so via the anaerobic oxidation of ammonium to dinitrogen gas, with nitrite as terminal electron acceptor. The first step in this so-called anammox reaction is the conversion of nitrite to nitric oxide by nitrite reductase. Next, nitric oxide is combined with ammonium to form hydrazine by hydrazine synthase, after which hydrazine is oxidized to dinitrogen gas by hydrazine dehydrogenase. In contrast to the other catabolic anammox enzymes, different anammox species encode different potential nitrite reductase enzymes. On top of that, there is a redundancy in genes encoding for nitrite reductase in single anammox species. The unusual diversity and redundancy in anammox nitrite reductases is unexplained. The genome of the model anammox species "Candidatus Kuenenia stuttgartiensis" encodes for three putative nitrite reductases. Here, we investigated which of these nitrite reductases is or are active in K. stuttgartiensis. Active nitric oxide-producing nitrite reductases were enriched from K. stuttgartiensis cells via fast protein liquid chromatography. Nitric oxide production by the enriched nitrite reductases was followed with membrane inlet mass spectrometry. Combining the activity assays with proteomics analysis indicated that the soluble nitrite reductases NirS and HAOr most strongly correlated with enzyme activity. This indicates that K. stuttgartiensis employs two distinct nitrite reductases to keep its nitric oxide pool replenished.

microbiology↗

bioRxiv2022

MS2Query: Reliable and Scalable MS2 Mass Spectral-based Analogue Search

Metabolomics-driven discoveries of biological samples remain hampered by the grand challenge of metabolite annotation and identification. Only few metabolites have an annotated spectrum in spectral libraries; hence, searching only for exact library matches generally returns a few hits. An attractive alternative is searching for so-called analogues as a starting point for structural annotations; analogues are library molecules which are not exact matches, but display a high chemical similarity. However, current analogue search implementations are not yet very reliable and relatively slow. Here, we present MS2Query, a machine learning-based tool that integrates mass spectral embedding-based chemical similarity predictors (Spec2Vec and MS2Deepscore) as well as detected precursor masses to rank potential analogues and exact matches. Benchmarking MS2Query on reference mass spectra and experimental case studies demonstrates an improved reliability and scalability. Thereby, MS2Query offers exciting opportunities for further increasing the annotation rate of complex metabolite mixtures and for discovering new biology.

bioinformatics↗