Biology subjects

Habenicht, T.

Publications and source records attributed to Habenicht, T..

4 recordsLinked to original sources

bioRxiv2025

Small subunits MttS and MttQ of the MttP transporter regulate trimethylamine transport in Methanosarcina mazei

Small proteins (<100 aa) have moved in the focus of science after being overlooked for decades due to bioinformatical and biochemical challenges. While mass spectrometry-coupled ribosome profiling of the mesophilic methanoarchaeon Methanosarcina mazei has recently unveiled a wealth of novel small ORFs, the functional roles of most of their products remain unknown. Here, we report the characterization of MttQ (98 aa) and MttS (49 aa), products of small ORFs situated in an operon alongside genes encoding a drug-metabolite-efflux (DME) family transporter (mttP) and other enzymes involved in trimethylamine (TMA) degradation. MttS and MttQ interact with MttP to form a stable oligomeric complex spanning the cytoplasmic membrane. TMA transport activity of the MttQ/MttS/MttP-complex is demonstrated via in vivo Escherichia coli cells heterologously expressing this system. Based on the reduced growth of a M. mazei mutant lacking mttS, on TMA as sole carbon source, we conclude that MttS governs the specificity of TMA transport. We posit that interactions between a DME transporter, e.g., MttP, and small proteins fueled evolution of the MttPQS complex and the advent of selective TMA uptake in methylotrophic methanoarchaea. These findings suggest an evolutionary mechanism on how small accessory proteins can alter conserved core functions in order to explore new ecological niches. Further, given that TMA levels in the human bloodstream influence cardiovascular disease risk but can be degraded by host-associated methanoarchaea containing a homolog of the MttPQS-complex, our findings present insight into an archaeal pathway with relevance to human health.

microbiology↗

bioRxiv2025

Small ORF16 modulates the Mtr complex activity in Methanosarcina mazei Goe1 depending on the molecular hydrogen availability

Until recently, small open reading frame (sORF)-encoded proteins of fewer than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in conventional detection methodologies. While numerous previously unannotated sORFs have recently been identified in the mesophilic archaeal model system Methanosarcina mazei, the physiological roles of most of their encoded small proteins remain unknown. We report here the functional characterization of sORF16 encoded small protein MtrR (49 amino acids) and show that it localizes oligomerically at the cytoplasmic membrane. There, it interacts with and influences the activity of tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex involved in energy metabolism. In vitro interaction and in vivo copurification assays revealed interactions between MtrR and the Mtr-complex, and microscale thermophoresis showed specific interactions with the MtrA subunit. Mutant strains lacking sORF16 exhibited significantly impaired growth in the presence of molecular hydrogen (H2), irrespective of the carbon source. We posit that by modulating the activity of the Mtr-complex, MtrR enables the archaeon adapt to changing environmental H2 conditions.

microbiology↗

bioRxiv2024

2-oxoglutarate triggers assembly of active dodecameric Methanosarcina mazei glutamine synthetase

Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 [A] cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on an allosteric binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

microbiology↗

bioRxiv2023

Small protein mediates inhibition of ammonium transport in Methanosarcina mazei - an ancient mechanism?

In the past decade, small open reading frames (sORFs) coding for proteins less than 70 amino acids (aa) in length have moved into the focus of Science. sORFs and corresponding small proteins have been recently identified in all three domains of life. However, the majority of small proteins remain functionally uncharacterized. While several bacterial small proteins have already been described, the number of identified and functionally characterized small proteins in archaea is still limited. In this study, we have discovered that the small protein 36 (sP36), which consists of only 61 aa, plays a critical role in regulating nitrogen metabolism in Methanosarcina mazei. The absence of sP36 significantly delays the growth of M. mazei when transitioning from nitrogen limitation to nitrogen sufficiency, as compared to the wild type. Through our in vivo experiments, we have observed that during nitrogen limitation, sP36 is dispersed throughout the cytoplasm; however, upon shifting the cells to nitrogen sufficiency, it relocates to the cytoplasmic membrane. Moreover, in vitro biochemical analysis clearly showed that sP36 interacts with high-affinity with the ammonium transporter AmtB1 present in the cytoplasmic membrane during nitrogen limitation, as well as with the PII-like protein GlnK1. Based on our findings, we propose that in response to an ammonium up-shift, sP36 targets the ammonium transporter AmtB1 and inhibits its activity by mediating the interaction with GlnK1. ImportanceSmall proteins containing fewer than 70 aa, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role for the small protein sP36 in the nitrogen metabolism of M. mazei, regulating the ammonium transporter AmtB1 according to nitrogen availability. This regulation might represent an ancient archaeal mechanism of AmtB1 inhibition by GlnK, in contrast to the well-studied regulation in bacteria, which depends on covalent modification of GlnK.

molecular biology↗