Biology subjects

de Kok, N. A. W.

Publications and source records attributed to de Kok, N. A. W..

4 recordsLinked to original sources

bioRxiv2024

Developing Hybrid Systems to Address O2 Uncoupling in Multi-Component Rieske Oxygenases

Rieske non-heme iron oxygenases (ROs) are redox enzymes that are essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful in applied enzymology due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H2O2) formation in the presence of a NADH cofactor regeneration system on the efficiency of CDO catalytic efficacy in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H2O2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h at a 3-fold increase in production rate compared to CDO-WT.

biochemistry↗

bioRxiv2023

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we resolve the functional mechanism of the antimicrobial action of the cationic tripeptide AMC-109 by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on the negatively charged plasma membrane of Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates with specificity for negatively charged membranes. Second, by incorporation into the S. aureus membrane the lateral membrane organization is affected, dissolving membrane nanodomains. Domain dissolution affects membrane functions such as protein sorting and cell wall synthesis, and is suggested to cause a loss of resistance of methicillin-resistant S. aureus (MRSA) to methicillin. As the AMC-109 mode of action is similar to the activity of the disinfectant benzalkonium chloride (BAK), a broad applicability, but with low cytotoxicity to human cells, is expected.

biophysics↗

bioRxiv2020

A promiscuous archaeal cardiolipin synthase generating a variety of cardiolipins and phospholipids

Cardiolipin (DPCL) biosynthesis has barely been explored in Archaeal isoprenoid-based ether lipid membranes. Here, we identified a cardiolipin synthase (MhCls) from the mesophilic anaerobic methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and subsequently characterized by LC-MS. MhCls utilizes two archaetidylglycerol molecules in a transesterification reaction to synthesize archaeal di-phosphate cardiolipin (aDPCL) and glycerol. The enzyme is invariant to the stereochemistry of the glycerol-backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol to generate di-phosphate cardiolipin (DPCL). Remarkably, in the presence of archaetidylglycerol and phosphatidylglycerol, MhCls formed an archaeal-bacterial hybrid di-phosphate cardiolipin (hDPCL), that so far has not been observed in nature. Due to the reversibility of the transesterification, cardiolipin can be converted back in presence of glycerol into phosphatidylglycerol. In the presence of other compounds that contain primary hydroxyl groups (e.g. alcohols, water, sugars) various natural and unique artificial phospholipid species could be synthesized, including multiple di-phosphate cardiolipin species. Moreover, MhCls could utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphate cardiolipin, emphasizing the promiscuity of this cardiolipin synthase.

molecular biology↗

bioRxiv2020

CRISPR-Based Transcriptional Activation Tool for Silent Genes in Filamentous Fungi

Filamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA "plug-and-play" module, into an AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

microbiology↗