Biology subjects

Leung, I. K. H.

Publications and source records attributed to Leung, I. K. H..

6 recordsLinked to original sources

bioRxiv2025

Redox-controlled dimerisation regulates ethylene biosynthesis

Ethylene is a central plant hormone that orchestrates growth, development, senescence, and stress responses. Because it is gaseous, ethylene must be synthesised on demand, yet the catalytic and regulatory mechanisms of its biosynthetic enzyme, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), remain poorly understood. Here, using structural, biophysical, and computational analyses, we uncovered two principles: ACO catalysis relies on an induced-fit mechanism, and disulfide-mediated dimerisation via a conserved cysteine acts as a redox switch toggling ACO between active monomer and inactive dimer. This previously unrecognised regulatory layer positions ACO as a redox sensor in plant cells, revealing a fundamental control point in ethylene biosynthesis. Given ethylenes pivotal role in crop productivity and stress resilience, these findings open new opportunities for precise manipulation of hormone signalling in agriculture and biotechnology.

biochemistry↗

bioRxiv2025

Decoding the Redox-Driven Fate of Organic Micropollutants through Microbial Co-metabolic and ROS-Mediated Degradation in Wastewater

The uncontrolled release of organic micropollutants (OMPs) from wastewater treatment plants highlights a failure to synergize co-metabolic and reactive oxygen species (ROS)-driven biodegradation pathways. Here, we demonstrate that engineered redox cycling--dynamic fluctuations between oxic and anoxic conditions--fundamentally reshapes microbial metabolism and OMP reaction networks to dramatically enhance removal. Using an integrated multi-omics approach centered on paired mass distance (PMD) reactomics, we show that redox cycling increased the removal of 32 diverse OMPs from a baseline of 32% up to 67%. This enhancement was driven by a complete restructuring of degradation mechanisms: cycling stimulated microbial amino acid and fatty acid metabolism by up to 42%, which coupled to controlled ROS production where oxidative pathways (+15.995 Da) accounted for 47% of transformations under intermittent aeration. We mapped distinct "enzymatic fingerprints," with strong correlations (r > 0.7) linking Proteobacteria monooxygenases to oxidative reactions and Rhodobacteraceae dehydrogenases to reductive ones (+2.016 Da), revealing clear functional specialization. Ultimately, the redox regime dictates the entire OMP transformation network topology, shifting pathways from simple hydroxylation to complex, multi-step networks. This work provides a mechanistic framework establishing redox manipulation as a powerful strategy to synergistically activate degradation pathways, allowing existing infrastructure to meet stringent discharge regulations.

bioengineering↗

bioRxiv2025

Dynamic allostery drives acetyl-CoA-mediated activation of Mycobacterium tuberculosis isocitrate lyase 2

Mycobacterium tuberculosis isocitrate lyase 2 (ICL2) is an allosterically regulated enzyme that enables the bacterium to survive on non-glycolytic substrates during infection. Previous studies showed that ICL2 is allosterically regulated by acetyl-CoA and its analogues but the molecular mechanism underpinning this regulation is unknown. Here, we use protein NMR, crystallography, molecular dynamics, and mutagenesis studies to show that two unique structural features of ICL2, its C-terminal domain and a unique helical substructure on its N-terminal catalytic domain, play important roles in the enzymes allostery. In particular, we found that the binding of acetyl-CoA promotes the dimerisation of the C-terminal domain and disrupts its interactions with the unique helical substructure on the N-terminal domain. This leads to conformational changes in the ICL2 enzyme that induces activation. Taken together, our findings reveal, for the first time, how the binding of acetyl-CoA, which is not an ICL2 substrate, induces ICL2 activation. By extension, the work also identifies a novel allosteric mechanism controlling M. tuberculosis metabolism that is amenable to therapeutic manipulation. Significance StatementMycobacterium tuberculosis isocitrate lyase 2 (ICL2) was previously shown to be activated by acetyl-CoA and propionyl-CoA - two central metabolites generated by the metabolism of sugars and fatty acids. However, it is not known how the binding of these metabolites leads to the activation of ICL2. Together with its isoform ICL1, ICL2 has been shown to be essential for the survival and pathogenesis of the bacterium. Understanding how this regulation occurs can help design novel treatments to target this protein and eradicate these bacteria, which cause the most deaths worldwide due to a single bacterial agent. This system also presents a fascinating model to examine allostery in proteins, with the techniques illustrated in this paper being applicable to other allosteric proteins.

biochemistry↗

bioRxiv2025

Protease mimicry: dissecting the ester bond crosslinking mechanics in bacterial adhesin proteins

The ester bond crosslink discovered within bacterial adhesin proteins offers a captivating insight into the convergent evolution of enzyme-like machinery. Crystal structures reveal a putative catalytic triad comprising an acid-base-nucleophile combination and an oxyanion-like site that suggest a serine protease-like mechanism drives the crosslinking process. We now provide confirmation of the mechanism, revealing functional catalytic dyads or triads, and the recapitulation of protease machinery from a Pseudomonas bacterium and a human cytomegalovirus related only by convergent evolution. Molecular dynamics simulations show how a conservative threonine-to-serine mutation of the nucleophile induces hydrolysis and eliminates the ester bond crosslink. Collectively, our structural, functional, and computational efforts detail the molecular intricacies of intramolecular ester bond formation and underscore the convergent evolutionary adaptations of bacteria in exploiting enzyme-like machinery to protect essential adhesin proteins from the mechanical, biological, and chemical hostilities of the bacterias replicative niche.

biochemistry↗

bioRxiv2024

Microbial Degradation of Contaminants of Emerging Concern by Pseudomonas putida in Wastewater Treatment Plants

Contaminants of Emerging Concern (CECs) are a class of contaminants that are commonly found in urban wastewater treatment plants (WWTP) at low concentrations. These compounds include antibiotic drugs, personal care products, and industrial chemicals, which are widely used in modern society. Although they provide undeniable benefits in our daily lives, their accumulation in the environment poses a significant risk to human health and the ecosystem. Due to the incomplete removal of these compounds by traditional WWTPs, there is a need to understand the interactions between microbes and CECs to develop effective solutions to this environmental challenge. Pseudomonas putida is a bacterium commonly found in water-related habitats, including freshwater streams, forests, and WWTPs. It is known for its ability to degrade various organic compounds, making it a suitable model for understanding the linkages between bacterial enzymes and CECs found in WWTPs. In this study, isolated Pseudomonas putida strain KT2440 was cultured in lab-scale reactors to mimic the WWTP environment. The degradation of this isolated strain towards different groups of CECs were studied over a 24-hour period using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Mass spectrometry-based quantitative proteomics was also performed at different sampling points to establish a linkage between the enzyme expression profile of this strain and its degradation of CECs via bioinformatics analysis. The results of this study demonstrated that Pseudomonas putida KT2440 is capable of degrading atrazine, acetamiprid, carbendazim, diclofenac acid, erythromycin, and sulfamethazine at lab scale. At the 20-hour time point, the enzyme expression profile indicated a high protein abundance of various oxidoreductases that could explain the observed CEC degradation.The studys findings provide important insights into the potential of Pseudomonas putida as a bioremediation agent for CECs in WWTPs. The degradation of CECs by Pseudomonas putida KT2440 is a significant contribution to the bioremediation process, and its enzyme expression profile can serve as a case study for more complex interactions of microbes and CECs in real WWTPs. Furthermore, this study supports the potential of Pseudomonas putida as a promising strain for various bioremediation applications. Finally, this studys results can help develop more efficient and effective methods for removing CECs from wastewater, which can contribute to the protection of public health and the environment.

microbiology↗

bioRxiv2024

Microbial Metabolic Enzymes, Pathways and Microbial Hosts for Co-Metabolic Degradation of Organic Micropollutants in Wastewater

Organic micropollutants (OMPs) in wastewater present significant environmental challenges, but effective removal strategies are hindered by our limited understanding of their co-metabolic biodegradation. We aim to elucidate the microbial enzymes, metabolic pathways, and community members involved in OMP co-metabolic degradation, thereby paving the way for more effective wastewater treatment strategies. We integrated multi-omics (metagenomics, metaproteomics, and metabolomics) and functional group analysis to investigate 24 OMPs under three aeration conditions. Our findings reveal that oxidoreductases, particularly cytochrome P450s and peroxidases, are crucial for recalcitrant OMPs containing halogen groups (-Cl, -F) like fluoxetine and diuron. Hydrolases, including amidases, are instrumental in targeting amide-containing (-CONH2) OMPs such as bezafibrate and carbamazepine. Regarding microbial metabolism involved in OMP co-metabolic degradation, we found that amino acid metabolism is crucial for degrading amine-containing (-NH2) OMPs like metoprolol and citalopram. Lipid metabolism, particularly for fatty acids, contributes to the degradation of carboxylic acid (-COOH) containing OMPs such as bezafibrate and naproxen. Finally, with Actinobacteria, Bacteroidetes, and Proteobacteria emerging as primary contributors to these functionalities, we established connections between OMP functional groups, degradation enzymes, metabolic pathways, and microbial phyla. Our findings provide generalized insights into structure-function relationships in OMP co-metabolic degradation, offering the potential for improved wastewater treatment strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/611302v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1785fd8org.highwire.dtl.DTLVardef@1dc732dorg.highwire.dtl.DTLVardef@d9eaborg.highwire.dtl.DTLVardef@2c2813_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗