Biology subjects

Ramanathan, G.

Publications and source records attributed to Ramanathan, G..

4 recordsLinked to original sources

bioRxiv2025

Bifunctional Architecture Enables Substrate Catalysis and Channeling in Paracoccus TMAO Demethylase

Substrate channeling enhances efficiency and prevents toxicity by directing unstable intermediates between active sites. Trimethylamine N-oxide demethylase (TDM) degrades trimethylamine N-oxide (TMAO) to dimethylamine and formaldehyde (HCHO), but the fate of HCHO has remained unclear. We report cryo-EM structures of TDM in apo, substrate-, and product-bound states that reveal a previously unknown channeling pathway. Combined structural, biochemical, and target molecular dynamics analyses show that HCHO is generated in a catalytic core and guided through a tunnel to a remote tetrahydrofolate (THF)-binding site, where it forms methylene-THF. Thus, TDM emerges as a bifunctional enzyme that unites TMAO demethylation with one-carbon transfer, providing a mechanistic explanation for its role in metabolic efficiency and detoxification.

biophysics↗

bioRxiv2025

Optimisation and validation of a rapid RP-HPLC method for budesonide quantification from polymeric nanoparticles with application to stability and drug release studies

Robust and sustainable analytical tools are essential for the design, manufacture, and evaluation of advanced drug delivery systems. This study reports the first validated reversed-phase high-performance liquid chromatography (RP-HPLC) method for quantifying budesonide encapsulated within poly(lactic-co-glycolic acid) (PLGA)-based, layer-by-layer (LbL) coated nanoparticles intended for colonic delivery in inflammatory bowel disease (IBD). Using response surface methodology with a central composite design, optimal conditions were identified as acetonitrile:water (80:20, v/v) under isocratic mode, achieving complete separation within 5 min at a flow rate of 0.34 mL/min and detection at 244 nm. The method is buffer-free, rapid, and solvent-efficient, resulting in a favorable greenness profile, further confirmed by GAPI and NEMI assessments. Validation in line with ICH Q2(R2) demonstrated excellent linearity (R{superscript 2} > 0.999), precision (%RSD < 2 %), accuracy, specificity, and sensitivity (LOD: 0.04 {micro}g/mL; LOQ: 1.2 {micro}g/mL). Forced degradation studies under acidic, alkaline, oxidative, thermal, photolytic, and photostatic conditions showed that the LbL coating markedly enhanced drug stability, particularly against thermal and photostatic stress, while >75% degradation occurred in alkaline and oxidative environments. In vitro release profiling under simulated gastrointestinal conditions demonstrated sustained, pH-responsive release (20.6% over 48 h), consistent with colonic targeting. This validated, green, and stability-indicating method integrates controlled release assessment with comprehensive performance evaluation, providing a versatile platform for quality control of nanoparticulate drug delivery systems and supporting their progression from formulation development to clinical translation

pharmacology and toxicology↗

bioRxiv2024

Optimising the viscoelastic properties of hyaluronic acid hydrogels through colloidal particle interactions: a response surface methodology approach

Enhancing the viscoelastic characteristics of hydrogel systems through strategic colloidal particle interactions is paramount for their functionality in rectal gel applications. This investigation delves into the synergistic interactions between cationic nanoparticles (CNPs), anionic nanoparticles (ANPs), and composite nanoparticles (NPs) within a hyaluronic acid (HA) hydrogel matrix, employing response surface methodology (RSM) for optimisation. Critical parameters, namely the volume fraction of NPs and oscillatory amplitude, were meticulously calibrated to achieve optimal complex viscosity, as determined by advanced rheometric analysis. The findings reveal substantial effects of CNPs, ANPs, and mixed NPs on the viscoelasticity of the HA hydrogel, with complex viscosity measurements of 490.82 {+/-} 10.57, 214.70 {+/-} 8.96, and 328.46 {+/-} 6.67 mPa{middle dot}s, respectively. The hydrogel system with mixed NPs exhibited a strong concordance with empirical data (R{superscript 2} = 0.9843), validating the predictive precision of the model. Morphological assessments uncovered a highly interconnected network within the HA gel-particle composite, characterised by both densely and sparsely packed porous architectures. This study presents a robust framework for modulating viscoelastic properties in colloidal particle-gel systems, providing pivotal insights for the development of advanced rectal gel formulations.

bioengineering↗

bioRxiv2019

Breaking a Strong Amide Bond: Structure and Properties of Dimethylformamidase.

Dimethylformamidase (DMFase) breaks down the human-made synthetic solvent N,N-dimethyl formamide(DMF) used extensively in industry(1). DMF is not known to exist in nature and was first synthesized in 1893. In spite of the recent origin of DMF, certain bacterial species such as Paracoccus, Pseudomonas, and Alcaligenes have evolved pathways to breakdown DMF and use them as carbon and nitrogen source for growth(2, 3). The work presented here provides a molecular basis for the ability of DMFase from Paracoccus to function in exacting conditions of high solvent concentrations, temperature and ionic strength to catalyze the hydrolysis of a stable amide bond. The structure reveals a multimeric complex of the 2{beta}2 type or (2{beta}2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed folds and as yet of unknown evolutionary origin. The active site is made of a distinctive mononuclear iron that is coordinated by two tyrosine residues and a glutamic acid residue. The hydrolytic cleavage of the amide bond is catalyzed at the Fe3+ site with a proximal glutamate probably acting as the base. The change in the quaternary structure is salt dependent with high salt resulting in the larger oligomeric state. Kinetic characterization reveals an enzyme that shows cooperativity between subunits and the structure provides clues on the interconnection between the active sites. Significance StatementN,N-dimethyl formamide(DMF) is a commonly used industrial solvent that was first synthesized in 1893. The properties that make DMF a highly desired solvent also makes it a difficult compound to breakdown. Yet, certain bacteria have evolved to survive in environments polluted by DMF and have enzymes that breakdown DMF and use it as their carbon and nitrogen source. The molecular structure of the enzyme that breaks down the stable amide bond in these bacteria, reveals two new protein folds and a unique mononuclear iron active site. The work reported here provides the structural and biochemical framework to query the evolutionary origins of the protein, as well as in engineering this enzyme for use in bioremediation of a human made toxic solvent.

biochemistry↗