Biology subjects

Snoep, J. L.

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

8 recordsLinked to original sources

bioRxiv2026

Story about honest mistakes: The cyanobacterium Synechocystis has a promiscuous Entner-Doudoroff (ED) aldolase but no functional ED pathway.

In 2016, the glycolytic Entner-Doudoroff (ED) pathway was reported in cyanobacteria and plants (1). The claim was based on the biochemical characterization of its key enzyme the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase also named ED aldolase (EDA), on protein sequence alignments, physiological data from cyanobacterial mutants, and the in vivo detection of an ED pathway specific metabolite (1). However, two enzymes 6-phosphogluconate (6PG) dehydratase (EDD) and EDA are unique to this route. A recent study suggests that EDD (Slr0452) from Synechocystis sp. PCC 6803 most likely encodes an enzyme involved exclusively in amino acid synthesis, indicating that a complete ED pathway would be missing (2). To address the presence or absence of the ED pathway in Synechocystis, we conducted extended biochemical and physiological studies, revisited old data and resolved contradictions. These investigations reveal that Synechocystis lacks both an ED pathway and a glucose dehydrogenase/glucokinase (GDH/GK) bypass but contains a promiscuous aldolase EDA. EDA prefers KDPG as substrate but also decarboxylates oxaloacetate (OAA) and cleaves 2-keto-4-hydroxyglutarate (KHG). Synthesis of KDPG from pyruvate and glyceraldehyde 3-phosphate (GAP) is catalyzed with very low efficiency. These in vitro data suggest that EDA might be involved in the phosphoenolpyruvate (PEP)-pyruvate-OAA node and proline catabolism, which requires further clarification. The previous misconception was based on missing enzymatic characterizations, the oversight of a secondary mutation in a deletion strain, and an outdated view on carbohydrate fluxes. We conclude with a list of lessons and provide a solid foundation for future investigations into the role of EDA in cyanobacteria and other photoautotrophs. Significance statementThis study provides a retrospective on why, for many years, it was mistakenly assumed that the glycolytic Entner-Doudoroff (ED) pathway exists in the cyanobacterium Synechocystis sp. PCC 6803. It shows that the first enzyme of this pathway, ED dehydratase EDD is absent, while the second enzyme, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase EDA, is present but is promiscuous, cleaving KDPG in addition to 2-keto-4-hydroxyglutarate (KHG) and decarboxylating oxaloacetate (OAA) in vitro. Finally, valuable lessons are drawn from prior misconceptions and experimental limitations. This study provides a solid foundation for future studies on the role of the ED aldolase in absence of the ED pathway in cyanobacteria and other photoautotrophs.

microbiology↗

bioRxiv2026

Inhibition of the androgen-activating enzyme AKR1C3 selectively decreases systemic and intra-adipose 11-oxygenated androgens in women

Androgen excess drives metabolic and reproductive complications in polycystic ovary syndrome (PCOS), affecting 10-15% of women globally. Aldo-keto reductase 1C3 (AKR1C3) converts inactive precursors from both the classic and the recently identified 11-oxygenated androgen pathways, generating testosterone and 11-ketotestosterone, respectively, which exert comparable androgen receptor activation. Both circulate in similar concentrations in premenopausal women while 11-ketotestosterone is predominant after menopause and in PCOS. Here, we show that adipocytes are a major site of AKR1C3 and androgen receptor expression, with increased expression in women and individuals with obesity. Using human female adipose tissue explants, we find a much higher activation of 11-oxygenated over classic androgens, observing a decrease in 11-oxygenated but not classic androgen activation by AKR1C3 inhibition. Correspondingly, we demonstrate that AKR1C3 inhibitor treatment in premenopausal women selectively disrupts the activation of 11-oxygenated androgens. Pharmacological targeting of AKR1C3 provides a novel strategy to alleviate systemic and intra-adipose 11-oxygenated androgen excess. One Sentence SummaryInhibition of the androgen-activating enzyme AKR1C3 results in a major decrease in 11-oxygenated but not classic androgens in women.

physiology↗

bioRxiv2025

Establishment of an efficient one-step enzymatic synthesis of cyclic-2,3-diphosphoglycerate

Extremolytes - unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their promise, only a few extremolytes, such as ectoine and hydroxyectoine, are commercially established, primarily due to the lack of efficient production strategies for other compounds. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg2+, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2 U mg-1, with KM values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 minutes, using just 0.5 U of recombinant MfcDPGS at 55{degrees}C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value-added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

bioengineering↗

bioRxiv2025

Fructose-1,6-bisphosphatase (FBPase) fine-tunes heterotrophic growth in cyanobacteria

Cyanobacteria switch their carbon metabolism between photoautotrophy and heterotrophy during diurnal cycles. In cyanobacteria, the classical glycolytic control point is characterized by two catabolic phosphofructokinases (PFKs) and a bifunctional anabolic fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (F/SBPase; slr2094) catalyzing two key reactions in the Calvin-Benson-Bassham (CBB) cycle. In addition, Synechocystis possesses a fructose-1,6-bisphosphatase (FBPase; slr0952) with yet unknown physiological function and biochemical properties. Hence, our aim was to investigate the FBPase and the interplay of the four enzymes in photoautotrophic and heterotrophic carbon metabolism. We discovered that FBPase is specific for FBP, showing no SBPase activity, and unlike F/SBPase does not exhibit any biochemical regulatory properties. Growth studies with deletion mutants revealed that FBPase and PFKs play a major role under heterotrophic conditions. In contrast to F/SBPase, FBPase is not involved in the CBB cycle, but instead fine-tunes heterotrophic growth. Transaldolase cannot replace the function of SBPase in the CBB cycle. In conclusion, the classical Embden-Meyerhoff-Parnass pathway control point, which is known to be mediated by the antagonistic enzyme pair PFK and FBPase in heterotrophic bacteria and eukaryotes, is also present in Synechocystis. We found redox-insensitive FBPases from plant chloroplasts to be closely related to Synechocystis FBPase, indicating that they might serve a similar function. HighlightSynechocystis fructose-1,6-bisphosphatase (slr0952) is unlike fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (slr2094) monofunctional, not redox-regulated, and supports heterotrophy in darkness, despite catalyzing an anabolic reaction. Thus, presumably Slr2094 alone drives two Calvin-Benson-Bassham cycle key reactions.

plant biology↗

bioRxiv2024

The archaeal family 3 polyphosphate kinase reveals a function of polyphosphate as energy buffer under low energy charge

Inorganic polyphosphate, a linear polymer of orthophosphate residues linked by phosphoanhydride bonds, occurs in all three domains of life and plays a diverse and prominent role in metabolism and cellular regulation. While the polyphosphate metabolism and its physiological significance have been well studied in bacteria and eukaryotes including human, there are only few studies in archaea available so far. In Crenarchaeota including members of Sulfolobaceae, the presence of polyphosphate and degradation via exopolyphosphatase has been reported and there is some evidence for a functional role in metal ion chelation, biofilm formation, adhesion and motility, however, the nature of the crenarchaeal polyphosphate kinase is still unknown. Here we used the crenarchaeal model organism Sulfolobus acidocaldarius to study the enzymes involved in polyphosphate synthesis. The two genes annotated as thymidylate kinase (saci_2019 and saci_2020), localized downstream of the exopolyphosphatase, were identified as the missing polyphosphate kinase in S. acidocaldarius (SaPPK3). Thymidylate kinase activity was confirmed for Saci_0893. Notably Saci_2020 showed no polyphosphate kinase activity on its own but served as regulatory subunit (rPPK3) and was able to enhance polyphosphate kinase activity of the catalytically active subunit Saci_2019 (cPPK3). Heteromeric polyphosphate kinase activity is reversible and shows a clear preference for polyP-dependent nucleotide kinase activity, i.e. polyP-dependent formation of ATP from ADP (12.4 U/mg) and to a lower extent of GDP to GTP whereas AMP does not serve as substrate. PPK activity in the direction of ATP-dependent polyP synthesis is rather low (0.25 U/mg); GTP was not used as phosphoryl donor. A combined experimental modelling approach using quantitative 31P NMR allowed to follow the reversible enzyme reaction for both ATP and polyP synthesis. PolyP synthesis was only observed when the ATP/ADP ratio was kept high, using an ATP recycling system. In absence of such a recycling system, all incubations with polyP and PPK would reach an equilibrium state with an ATP/ADP ratio between 3 and 4, independent of the initial conditions. Structural and sequence comparisons as well as phylogenetic analysis reveal that the S. acidocaldarius PPK is a member of a new PPK family, named PPK3, within the thymidylate kinase family of the P-loop kinase superfamily, clearly separated from PPK2. Our studies show that polyP, in addition to its function as phosphate storage, has a special importance for the energy homeostasis of S. acidocaldarius and due to its reversibility serves as energy buffer under low energy charge enabling a quick response to changes in cellular demand.

microbiology↗

bioRxiv2024

De novo synthesis of fatty acids in Archaea via an archaeal fatty acid synthase complex

Archaea synthesize membranes using isoprenoid-based ether lipids, whereas Bacteria and Eukarya use fatty acid-based ester lipids. While the factors responsible for this "lipid divide" remain unclear, this has important implications for understanding the evolutionary history of eukaryotes, which likely originated from within the Archaea and therefore changed membrane composition from isoprenoid-based to fatty acid-based lipids. Here, using 13C labelling studies, we demonstrate that the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii are capable of de novo fatty acid synthesis. Biochemical characterization and in vitro pathway reconstitution identify the key enzymes of a newly proposed fatty acid synthesis pathway in S. acidocaldarius and show that ketothiolase, ketoacyl-CoA reductase, and hydroxyacyl-CoA dehydratase form a stable assembly mediated by a DUF35 domain protein, which represents the first characterization of an archaeal fatty acid synthase complex. The final step is catalysed by an NADPH-dependent enoyl-CoA reductase. Deletion of the enoyl-CoA reductase demonstrate that this pathway operates in vivo in S. acidocaldarius. The presented results including phylogenetic analysis reveal that the potential to synthesize fatty acids is widespread across archaeal lineages. Collectively, our findings demonstrate that archaea are capable of synthesizing fatty acids, elucidate the molecular mechanisms involved in this process and provide additional insights into the evolutionary histories of fatty acid synthesis in archaea.

microbiology↗

bioRxiv2022

Cyclin CLB2 mRNA localization determines efficient protein synthesis to orchestrate bud growth and cell cycle progression

Clb2 is a conserved B-type cyclin essential for mitotic progression in Saccharomyces cerevisiae, with expression tightly regulated at transcriptional and proteolytic levels. However, it remains unclear whether Clb2 protein synthesis is regulated and responsive to cell growth. Here, we show that CLB2 mRNA localizes to the bud via the She2/She3 complex, while Clb2 protein accumulates in the mother nucleus. This mRNA localization enhances translation without affecting protein localization. A structured RNA element, a ZIP-code, is located within the coding sequence and is required, but not sufficient, for both mRNA transport and protein expression. Mutation of this ZIP code disrupts mRNA localization, reduces Clb2 synthesis, increases budded phase duration and daughter cell size. In wild-type cells, Clb2 protein levels scale with bud growth, a coupling lost in ZIP code mutants. These findings reveal a mechanism by which mRNA localization and translation are coordinated to link cell growth with cell cycle progression.

cell biology↗

bioRxiv2018

Harmonizing semantic annotations for computational models in biology

Life science researchers use computational models to articulate and test hypotheses about the behavior of biological systems. Semantic annotation is a critical component for enhancing the interoperability and reusability of such models as well as for the integration of the data needed for model parameterization and validation. Encoded as machine-readable links to knowledge resource terms, semantic annotations describe the computational or biological meaning of what models and data represent. These annotations help researchers find and repurpose models, accelerate model composition, and enable knowledge integration across model repositories and experimental data stores. However, realizing the potential benefits of semantic annotation requires the development of model annotation standards that adhere to a community-based annotation protocol. Without such standards, tool developers must account for a variety of annotation formats and approaches, a situation that can become prohibitively cumbersome and which can defeat the purpose of linking model elements to controlled knowledge resource terms. Currently, no consensus protocol for semantic annotation exists among the larger biological modeling community. Here, we report on the landscape of current semantic annotation practices among the COmputational Modeling in BIology NEtwork (COMBINE) community and provide a set of recommendations for building a consensus approach to semantic annotation.

scientific communication and education↗