Biology subjects

Harmer, N.

Publications and source records attributed to Harmer, N..

4 recordsLinked to original sources

bioRxiv2025

Establishment of an efficient one-step enzymatic synthesis of cyclic-2,3-diphosphoglycerate

Extremolytes - unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their promise, only a few extremolytes, such as ectoine and hydroxyectoine, are commercially established, primarily due to the lack of efficient production strategies for other compounds. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg2+, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2 U mg-1, with KM values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 minutes, using just 0.5 U of recombinant MfcDPGS at 55{degrees}C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value-added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

bioengineering↗

bioRxiv2021

Structure and function of N-acetylglucosamine kinase illuminates the catalytic mechanism of ROK kinases

N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolise environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase NagK performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Plesiomonas shigelloides in complex with GlcNAc and the ATP analogue AMP-PNP. Surprisingly, PsNagK showed two conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Molecular dynamics modelling of catalytic ion binding confirmed the location of the essential catalytic metal. Site-directed mutagenesis confirmed the catalytic base, and that the metal coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase.

biochemistry↗

bioRxiv2019

Drug screening to identify compounds to act as co-therapies for the treatment of pathogenic Burkholderia

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics, and is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to most currently available antibiotics. The most efficacious treatment for melioidosis requires at least two weeks of intravenous treatment with ceftazidime or meropenem. This places a large treatment burden on the predominantly middle income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z factor of 0.68. We screened a library of 61,250 compounds, and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six \"best in class\" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, preliminary data also demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy against these bacteria.

microbiology↗

bioRxiv2019

Engineering a seven enzyme biotransformation using mathematical modelling and characterized enzyme parts

Multi-step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi-step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi-step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.\n\nSignificance statementThis study examines the challenge of modeling and optimizing multi-enzyme cascades. We detail the development, testing and optimization of a deterministic model of a three enzyme cascade with four cofactor regeneration enzymes. Significantly, the model could be easily used to predict the optimal concentrations of each enzyme in order to get maximum flux through the cascade. This prediction was strongly validated experimentally. The success of our model demonstrates that robust models of systems of at least seven enzymes are readily achievable. We highlight the importance of following good modeling practice to evaluate model quality and limitations. Examining deviations from expected behavior provided additional insight into the model and enzymes. This work provides a template for developing larger deterministic models of enzyme cascades.

biochemistry↗