Inhibitory Evaluation of αPMM/PGM from <i>Pseudomonas aeruginosa</i>: Chemical Synthesis, Enzyme Kinetics, and Protein Crystallographic Study

Zhu, Jian‐She; Stiers, Kyle M.; Soleimani, Ebrahim; Groves, Brandon R.; Beamer, Lesa J.; Jakeman, David L.

doi:10.1021/acs.joc.9b01305

Cited by 8 publications

(12 citation statements)

References 37 publications

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“…To assess the effects of ligand on thermal denaturation, the substrate analog glucopyranosyl‐1‐methyl‐phosphonic acid (G1CP) was added to a final concentration of 4 mM. G1CP was prepared as previously described …”

Section: Methodsmentioning

confidence: 99%

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1

Stiers

Hansen

Daghlas

et al. 2020

J of Inher Metab Disea

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Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease‐related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild‐type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. Synopsis Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability.

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Section: Methodsmentioning

confidence: 99%

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1

Stiers

Hansen

Daghlas

et al. 2020

J of Inher Metab Disea

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“…S7A). Previous work has successfully demonstrated the use of substituted ketoheptoses and other phosphosugar analogues as inhibitors of microbial phosphomannomutases (74,75), which we sought to replicate with HAD5. Using a panel of 11 phosphosugar analogues (Fig.…”

Section: Had5 Is Distinct From Human Pmms and Can Be Specifically Inhibitedmentioning

confidence: 99%

“…5A) suggests that the inhibitor binding sites have differences that may account for this. Compounds D1-D11 were synthesized as substrate mimetics that inhibit enzymes at the substrate binding pocket, which is supported by co-crystal structures with structurally related inhibitors bound to the active site of related proteins (75). We therefore hypothesized that D9 inhibits HAD5 at the active site.…”

Section: Had5 Is Distinct From Human Pmms and Can Be Specifically Inhibitedmentioning

confidence: 99%

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites

Frasse

Miller

Polino

et al. 2022

Journal of Biological Chemistry

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“…S7A). Previous work has successfully demonstrated the use of substituted ketoheptoses and other phosphosugar analogues as inhibitors of microbial phosphomannomutases (72,73), which we sought to replicate with HAD5. Using a panel of 11 phosphosugar analogues (Fig.…”

Section: Had5 Is Distinct From Human Pmms and Can Be Specifically Inhibitedmentioning

confidence: 99%

“…Synthesis of compounds D1-D11 (Fig. S8A) have been described previously (72,73), with the exception of compound D9, whose synthesis is described, and characterization data included, in the attached supporting information. To assess inhibition of HAD5, compounds D1-D11 were suspended in water and added to the phosphoglucomutase assay of HAD5 activity (the phosphomannomutase assay was not used, as cross inhibition was seen with downstream components of that assay, but not with the phosphoglucomutase assay; Fig S8D).…”

Section: Inhibition Assays Of Recombinant Had5mentioning

confidence: 99%

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria parasites

Frasse¹,

Miller

Soleimani

et al. 2021

Preprint

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The malaria parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of GPI anchor biosynthesis, the putative phosphomannomutase (EC 5.4.2.8) of the parasites, HAD5 (PF3D7_1017400). We confirm the phosphomannomutase and phosphoglucomutase activity of purified recombinant HAD5. By regulating expression of HAD5 in transgenic parasites, we demonstrate that HAD5 is required for malaria parasite egress and erythrocyte reinvasion. Finally, we determine the three-dimensional crystal structure of HAD5 and identify a substrate analog that specifically inhibits HAD5, compared to orthologous human phosphomannomutases. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition, and that HAD5 has potential as a multi-stage antimalarial target.

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Inhibitory Evaluation of αPMM/PGM from Pseudomonas aeruginosa: Chemical Synthesis, Enzyme Kinetics, and Protein Crystallographic Study

Cited by 8 publications

References 37 publications

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria parasites

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