Biology subjects

Hjorleifsson, J. G.

Publications and source records attributed to Hjorleifsson, J. G..

4 recordsLinked to original sources

bioRxiv2026

Structure-based virtual screening reveals new metabolite interactions and modulators of human glucose-6-phosphate isomerase

Exploring the ability of metabolic enzymes to interact with diverse small molecules can provide insights into their ligand recognition and activity regulation. Glucose-6-phosphate isomerase (GPI) is an enzyme that catalyzes the second step of glycolysis, a critical node that links glucose metabolism to other cytosolic pathways, such as pentose phosphate and hexosamine biosynthetic pathways. Despite its importance in cellular metabolism, little is known about the broad ligandability of GPI. Here, we performed a structure-based virtual screening using molecular docking to identify new modulators predicted to engage with the human GPI active site. Enzyme kinetic and binding studies confirmed eleven compounds as active-site binders of GPI, exhibiting inhibitory activities and binding affinities in upper micromolar to sub-millimolar range. The binding of several ligands to GPI active site was further supported by X-ray crystallographic data. In addition, in vitro assays were performed to evaluate the modulatory effects of the GPI hits on cellular glycolysis and viability. The identified inhibitors included the mono-phosphorylated metabolite of ribavirin, which inhibited glycolytic flux and cell viability; two compounds with phosphate bioisosteres; and endogenous phosphate-containing metabolites from the pentose phosphate, hexosamine, nucleotide, and mevalonate pathways. Collectively, these findings expanded the repertoire of human GPI ligands and revealed previously unrecognized GPI-metabolite interactions that may contribute to modulation of glycolytic flux.

biochemistry↗

bioRxiv2024

Weak Modulation of Human Glycolysis Isomerases by Metabolites: A Screening Study

Modulation of enzyme activity by metabolites represents the most efficient and rapid way of controlling metabolism. Investigating enzyme-metabolite interactions can deepen our understanding of metabolic control and aid in identifying enzyme modulators with potential therapeutic applications. These interactions vary in strength, with dissociation constants (Kd) ranging from strong (nM) to weak ({micro}M-mM). However, weak interactions are often overlooked due to the challenges in studying them. Despite this, weak modulators can reveal novel binding modes and serve as starting points for compound optimization. In this study, we aim to identify metabolites that weakly modulate the activity of human glucose-6-phosphate isomerase (GPI) and triosephosphate isomerase (TPI), which are potential therapeutic targets in tumor glycolysis. Through a combination of activity and binding assays, the screening revealed multiple weak inhibitors for the two targets, causing partial attenuation of their activity, with Kd and Ki in the low mM range. X-ray crystallography revealed six orthosteric ligands binding to the active sites - four inhibitors of GPI and two of TPI. Our findings underscore the role of weak interactions in enzyme regulation and may provide structural insights that could guide the design of inhibitors targeting human GPI and TPI in cancer intervention. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=45 SRC="FIGDIR/small/610356v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@574540org.highwire.dtl.DTLVardef@1837c4forg.highwire.dtl.DTLVardef@15661c8org.highwire.dtl.DTLVardef@2db0ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Enzyme stability and function can be affected by various environmental factors, such as temperature, pH and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0 - 1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small angle X-ray scattering (SAXS) showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

biochemistry↗

bioRxiv2020

High catalytic rate of the cold-active Vibrio alkaline phosphatase depends on a hydrogen bonding network involving a large interface loop

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part of cold-adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large-loop, that hovers over the active site of the other monomer. It presumably has a role in VAP high catalytic efficiency that accompanies extremely low thermal stability. We designed several different mutagenic variants of VAP with the aim of removing inter-subunit interactions at the dimer interface. Breaking the inter-subunit contacts from one residue in particular (Arg336) caused diminished temperature stability of the catalytically potent conformation and a drop in catalytic rate by a half. The relative B-factors of the R336L crystal structure, compared to the wild-type, confirmed increased surface flexibility in a loop on the opposite monomer, but not in the large-loop. Contrary to expectations, the observed reduction in stability with an expected increase in dynamic mobility resulted in reduced catalytic rate. This contradicts common theories explaining high catalytic rates of enzyme from cold-adapted organisms as being due to reduced internal cohesion bringing increased dynamic flexibility to catalytic groups. The large-loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl-group affinity (inactive).

biochemistry↗