Biology subjects

Bedi, R. K.

Publications and source records attributed to Bedi, R. K..

4 recordsLinked to original sources

bioRxiv2025

Rewiring Catalytic Craters: A Path for Engineering β-Glucosidases to Improve Glucose Tolerance

Bioethanol, a sustainable alternative to fossil fuels, is produced from cellulose via enzymatic saccharification. {beta}-Glucosidase, a key enzyme in this process, hydrolyses disaccharides into glucose but is limited by feedback inhibition. This study investigated GH1 {beta}-glucosidase from a soil metagenome (UnBGl1) to understand and overcome this limitation. We solved the near-atomic resolution crystal structure of UnBGl1 at 1.15 [A] as an apo enzyme and report the first high-resolution crystal structures of the enzyme in its pre-hydrolytic state as a cellobiose complex and covalent intermediate-bound state, capturing key stages of its catalytic mechanism. Structural analysis revealed three crucial glucose-binding subsites in UnBGl1s crater. Glucose binding at the -1 subsite induced a 1.4 [A] shift in E370, increasing the distance between catalytic glutamates and reducing enzymatic activity. The C188V variant, generated by rational engineering, significantly improved glucose tolerance to 2.5 M. Additionally, the H261W mutation at the +2 subsite enhanced kinetic properties by improving cellobiose affinity (Km = 22.87 {+/-} 1.1 mM) and shifted the optimal pH to 5.5 from 6.0. Comparative structural analysis with other glucose-tolerant GH1 {beta}-glucosidases revealed conserved residues at the -1 subsite crucial for substrate stabilization and +1 subsite residues interacting with glucose, offering targets for further optimization. Engineered UnBGl1 variants retained high stability and activity on sugarcane bagasse, demonstrating their potential for industrial cellulase cocktails. These findings provide a robust framework for engineering {beta}-glucosidases with enhanced glucose tolerance and catalytic efficiency, paving the way for improved bioethanol production and contributing to sustainable energy solutions.

biochemistry↗

bioRxiv2023

The catalytic mechanism of the RNA methyltransferase METTL3

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on mRNA in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a bisubstrate analogue representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release catalysed by METTL3, and suggests that the latter step is rate-limiting. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors. One sentence summaryA combined structural, biochemical, and computational approach reveals two distinct binding conformations of adenosine, the transition state of methyl transfer, and the catalytic mechanism of the human m6A RNA methyltransferase METTL3-14.

biochemistry↗

bioRxiv2020

METTL3 inhibitors for epitranscriptomic modulation of cellular processes

The methylase METTL3 is the writer enzyme of the N6-methyladenosine (m6A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor (UZH1a) with potency in a biochemical assay of 280 nM, while its enantiomer UZH1b is 100 times less active. The crystal structure of the complex of METTL3 with UZH1a illustrates the interactions that make it selective against protein methyltransferases. We observed a dose-dependent reduction in m6A methylation level of mRNA in several cell lines treated with UZH1a already after 16 h of exposure, as determined by triple-quadrupole LC mass spectrometry, while its enantiomer UZH1b was essentially inactive at concentrations up to 100 {micro}M. Interestingly, the kinetics of m6A level reduction in mRNAs followed a first-order reaction model, with a half-decay time {tau} of 1.8 h and a maximum m6A inhibition level of 70%, which is in line with the previously observed shorter half-life of m6A-modified mRNAs. Notably, treatment with the compounds did not alter cellular METTL3 levels, ruling out indirect effects on m6A levels. The effect of the m6A level depletion by UZH1a directly translated into growth inhibition of MOLM-13 leukemia cells, under short-term and long-term culture. Incubation of the MOLM-13 cells with UZH1a, but not with UZH1b, resulted in increased cell apoptosis and cell cycle arrest already after 16 h of incubation. Interestingly, other cell lines sensitive to METTL3 level (U2Os, HEK293T) did not reveal statistically significant differences between UZH1a and UZH1b in a cell viability assay, confirming that the degree of reliance on m6A signalling for survival can vary between cancers/cell types.

biochemistry↗

bioRxiv2019

Flexible binding of m6A reader protein YTHDC1 to its preferred RNA motif

N6-methyladenosine (m6A) is the most prevalent chemical modification in human mRNAs. Its recognition by reader proteins enables many cellular functions, including splicing and translation of mRNAs. However, the binding mechanisms of m6A-containing RNAs to their readers are still elusive due to the unclear roles of m6A-flanking ribonucleotides. Here, we use a model system, YTHDC1 with its RNA motif 5-G-2G-1(m6A)C+1U+2-3, to investigate the binding mechanisms by atomistic simulations, X-ray crystallography, and isothermal titration calorimetry. The experimental data and simulation results show that m6A is captured by an aromatic cage of YTHDC1 and the 3 terminus nucleotides are stabilized by cation-{pi}-{pi} interactions, while the 5 terminus remains flexible. Moreover, simulations of unbound RNA motifs reveal that the methyl group of m6A and the 5 terminus shift the conformational preferences of the oligoribonucleotides to the bound-like conformation, thereby facilitating the association process. The proposed binding mechanisms may help in the discovery of chemical probes against m6A reader proteins.

biophysics↗