Biology subjects

Richards, N.

Publications and source records attributed to Richards, N..

5 recordsLinked to original sources

bioRxiv2026

DNA Ligases Discriminate Between Natural and Non-Natural Base Pairs

Artificially Expanded Genetic Information Systems (AEGIS) increase the information content of nucleic acids by including new nucleobase pairings that are orthogonal to those of canonical Watson-Crick nucleobases. DNA ligases do not form direct interactions with the nucleobases during catalytic turnover, suggesting that these enzymes should efficiently and faithfully join double-stranded AEGIS substrates. Here we report the systematic investigation into the validity of this hypothesis for structurally-diverse DNA ligases employing substrates built from the eight nucleotide hachimoji genetic alphabet, where orthogonality is achieved by rearranging the hydrogen bonding patterns seen in canonical Watson-Crick pairs. We find that single, or multiple, non-canonical bases are well tolerated at the 5-end of the nick. However, tracts of consecutive non-canonical bases at the 3-end of the break significantly decrease ligation efficiency or abolish it altogether. Possible reasons for this apparent bias against non-canonical nucleobases could include incompatibility in electrostatic interactions between the ligase active site and the non-canonical substrates or altered conformational preferences and/or dynamics in key catalytic intermediates. We also observe single hachimoji mismatches are ligated more frequently than mis paired canonical bases, potentially due to promiscuous pairing of tautomeric forms of the non-canonical bases.

biochemistry↗

bioRxiv2023

Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase

How motions in enzymes might be linked to catalytic function is of considerable general interest. Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we use 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) to identify a functional role for the Arg-142 side chain as a gate that mediates ammonia access to a catalytically relevant intramolecular tunnel. Our 3DVA-derived hypothesis is assessed experimentally, using the R142I variant in which Arg-142 is replaced by isoleucine, and by molecular dynamics (MD) simulations on independent, computational models of the WT human ASNS monomer and its catalytically relevant, ternary complex with {beta}-aspartyl-AMP and MgPPi. Residue fluctuations in the MD trajectories for the human ASNS monomer are consistent with those determined for 3DVA-derived structures. These MD simulations also indicate that the gating function of Arg-142 is separate from the molecular events that form a continuous tunnel linking the two active sites. Experimental support for Arg-142 playing a role in intramolecular ammonia translocation is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS. MD simulations of computational models for the R142I variant and the R142I/{beta}-aspartyl-AMP/MgPPi ternary complex provide a possible molecular basis for this observation. Overall, the combination of 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

molecular biology↗

bioRxiv2021

Genomics analysis of Drosophila sechellia response to Morinda citrifolia fruit diet

Drosophila sechellia is an island endemic host specialist that has evolved to consume the toxic fruit of Morinda citrifolia, also known as noni fruit. Recent studies by our group and others have examined genome-wide gene expression responses of fruit flies to individual highly abundant compounds found in noni responsible for the fruits unique chemistry and toxicity. In order to relate these reductionist experiments to the gene expression responses to feeding on noni fruit itself, we fed rotten noni fruit to adult female D. sechellia and performed RNA-sequencing. Combining the reductionist and more wholistic approaches, we have identified candidate genes that may contribute to each individual compound and those that play a more general role in response to the fruit as a whole. Using the compound specific and general responses, we used transcription factor prediction analyses to identify the regulatory networks and specific regulators involved in the responses to each compound and the fruit itself. The identified genes and regulators represent the possible genetic mechanisms and biochemical pathways that contribute to toxin resistance and noni specialization in D. sechellia.

genomics↗

bioRxiv2020

Structure of the ForT/PRPP complex uncovers the mechanism of C-C bond formation in C-nucleotide antibiotic biosynthesis

C-C bond formation is at the heart of anabolism and organic chemistry, but relatively few enzymatic strategies for catalyzing this reaction are known. The enzyme ForT catalyzes C-C bond formation between 5-phosphoribosyl-1-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of the C-nucleotide formycin A 5-phosphate; we now report the 2.5 [A] resolution structure of the ForT/PRPP complex and thus locate the active site. Site-directed mutagenesis has identified those residues critical for PRPP recognition and catalysis. Structural conservation with GHMP kinases suggests that stabilization of the negatively charged pyrophosphate leaving group is crucial for catalysis in ForT. A mechanism for this new class of C-C bond forming enzymes is proposed. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/009662v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1817d54org.highwire.dtl.DTLVardef@4b3558org.highwire.dtl.DTLVardef@19b248eorg.highwire.dtl.DTLVardef@7d0c62_HPS_FORMAT_FIGEXP M_FIG C_FIG A new class of enzymes catalyse C-C bond formation by irreversible CO2 and pyrophosphate production.

biochemistry↗

bioRxiv2018

Molecular basis of human asparagine synthetase inhibitor specificity

Expression of the enzyme human asparagine synthetase (ASNS) promotes metastatic progression in breast cancer, which affects L-asparagine levels and tumor cell invasiveness. Human ASNS has therefore emerged as a bona fide drug target for cancer therapy. We have reported a slow-onset, tight binding ASNS inhibitor with nanomolar affinity, but our compound exhibits poor cell permeability. On the other hand, we show here that this inhibitor exhibits remarkable selectivity for the human ASNS in HCT-116 cell lysates. By determining the first high-resolution (1.85 [A]) X-ray crystal structure for human ASNS, we have built a computational model of the enzyme complexed to our inhibitor, which provides the first insights into the intermolecular interactions mediating specificity. These findings should facilitate the development of a second generation of ASNS inhibitors, leading to the discovery of drugs to prevent metastasis.

biochemistry↗