Biology subjects

Harmer, N. J.

Publications and source records attributed to Harmer, N. J..

6 recordsLinked to original sources

bioRxiv2025

Cryo-EM reveals multiple mechanisms of ribosome inhibition by doxycycline

Antimicrobial resistance is driving the search for new antibiotics and a greater understanding of their mechanism of action. Doxycycline is amongst the most-prescribed antimicrobials. It demonstrates a particularly low minimum inhibitory concentration against the zoonotic pathogen Coxiella burnetii. Doxycycline canonically targets the bacterial ribosome by blocking tRNA binding at the decoding centre (A-site) of the small subunit. Using cryo electron-microscopy, we analysed doxycycline binding to C. burnetii and Escherichia coli ribosomes. Both structures reveal unexpected binding at the exit tunnel in the large subunit. In C. burnetii three doxycycline molecules stack to block the tunnel. In E. coli one doxycycline molecule triggers a major uncharacterised conformation of the ribosome. This fundamentally reorganises the peptidyl transferase centre and blocks tRNA binding, challenging the concept that this region is largely static. We identify a new ribosomal protein in the C. burnetii large subunit and characterise an additional member of the prokaryotic ribosome hibernation promoting factor family. These insights into ribosome function and antibiotic action may aid the development of new ribosome inhibitor antibiotics.

biochemistry↗

bioRxiv2025

Structure and catalytic mechanism of methylisocitrate lyase, a potential drug target against Coxiella burnetii

We present a comprehensive investigation into the catalytic mechanism of methylisocitrate lyase, a potential drug target candidate against the zoonotic pathogen Coxiella burnetii, the causative agent of Q fever and a federal select agent. Current treatment regimens are prolonged, often with incomplete clearance of the pathogen. We utilised a structure-based bioinformatics pipeline to identify methylisocitrate lyase as a candidate therapeutic target against C. burnetii from a list of essential genes. Wild-type C. burnetii methylisocitrate lyase has a kcat of 32,000 s-1 (compared to 105 s-1 for Salmonella enterica) and isocitrate inhibits with a KI of 6 mM. We have determined the previously uncharacterised substrate-bound structure of this enzyme family, alongside product and inhibitor-bound structures. These structures of wild-type enzyme reveal that in the active state the catalytic C118 is positioned 2.98 [A] from O5 of methylisocitrate and Arg152 moves towards the substrate relative to the inhibitor bound structure. Analysis of structure-based mutants reveals that Arg152 and Glu110 are both essential for catalysis. We suggest that Arg152 acts as the catalytic base that initiates the methylisocitrate lyase reaction. These results deepen our understanding of the catalytic mechanism of methylisocitrate lyase and could aid the development of new therapeutics against C. burnetii.

biochemistry↗

bioRxiv2022

Survivor bias drives overestimation of stability in reconstructed ancestral proteins

Ancestral sequence reconstruction has been broadly employed over the past two decades to probe the evolutionary history of life. Many ancestral sequences are thermostable, supporting the "hot-start" hypothesis for lifes origin. Recent studies have observed thermostable ancient proteins that evolved in moderate temperatures. These effects were ascribed to "consensus bias". Here, we propose that "survivor bias" provides a complementary rationalisation for ancestral protein stability in alignment-based methods. As thermodynamically unstable proteins will be selected against, ancestral or consensus sequences derived from extant sequences are selected from a dataset biased towards the more stabilising amino acids in each position. We thoroughly explore the presence of survivor bias using a highly parameterizable in silico model of protein evolution that tracks stability at the population, protein, and amino acid levels. We show that ancestors and consensus sequences derived from populations evolved under selective pressure for stability throughout their history are significantly biased toward thermostability. Our work proposes a complementary explanation of the origin of thermostability in the burgeoning engineering tools of ancestral sequence reconstruction and consensuses. It provides guidance for the thorough derivation of conclusions from future ancestral sequence reconstruction work.

evolutionary biology↗

bioRxiv2021

Spinning sugars in antigen biosynthesis: a direct study of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii respectively. Streptose forms the central moiety of the antibiotic streptomycin, whilst DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalysed by the enzymes RmlA/RmlB/RmlC/RmlD. Streptose and DHHS biosynthesis unusually require a ring contraction step that might be performed by the orthologues of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii proposed the StrM and CBU1838 proteins respectively as RmlC orthologues. Here, we demonstrate through both coupled and direct observation studies that both enzymes can perform the RmlC 3,5 double epimerisation activity; and that this activity supports TDP-rhamnose biosynthesis in vivo. We demonstrate that proton exchange is faster at the 3 position than the 5 position, in contrast to a previously studied orthologue. We solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to previously characterised enzymes. These results further support the hypothesis that streptose and DHHS are biosynthesised using the TDP pathway and are consistent with the ring contraction step being performed on a double epimerised substrate, most likely by the RmlD paralogue. This work will support the determination of the full pathways for streptose and DHHS biosynthesis.

biochemistry↗

bioRxiv2020

Assessments of hepatitis B virus-like particles and Crm197 as carrier proteins in melioidosis glycoconjugate vaccines

The Tier 1 select agent Burkholderia pseudomallei is the causative agent of melioidosis, a global pathogen and a major cause of pneumonia and sepsis for which no licensed vaccines currently exist. Previous work has shown the potential for Burkholderia capsular polysaccharide (CPS) to be used as a vaccine antigen but the T-cell independent nature of the immune response to this molecule requires the use of this polysaccharide as a glycoconjugate for vaccination. Recent studies have focussed on the use of Crm197 (a non-toxic mutant protein derived from diphtheria toxin) as the carrier but there are concerns regarding its potential to cause interference with other vaccines containing Crm197. Therefore research with alternative carrier proteins would be beneficial. In this study, CPS was isolated from the non-pathogenic B. thailandensis strain E555. This was chemically conjugated to Crm197, or Tandem Core virus-like particles (TCVLP) consisting of hepatitis B core protein, which is the first documented use of VLPs in melioidosis vaccine development. Analysis of CPS-specific IgG antibody titres showed that mice vaccinated with the Crm197 conjugate generated significantly higher titres than the mice that received TCVLP-CPS but both conjugate vaccines were able to protect mice against intraperitoneal B. pseudomallei strain K96243 challenges of multiple median lethal doses.

immunology↗

bioRxiv2019

A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.

biochemistry↗