Characterization and functional insights into the Entamoeba histolytica pyridoxal kinase, an enzyme essential for its survival

Tarique, K.F.; Devi, S. Uma; Tomar, Priya; Ali, Mohammad Farhan; Rehman, S.A. Abdul; Gourinath, S.

doi:10.1016/j.jsb.2020.107645

Cited by 6 publications

(8 citation statements)

References 53 publications

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“…Notable structural differences from human PLK in the active site region of this enzyme were observed which means targeting this enzyme would be detrimental and affect the activity of all PLP-dependent enzymes of this human pathogen. In continuation of our previous work which showed EhPLK to be necessary for survival and growth of E. histolytica (Tarique et al, 2020), we have gone further to identify two novel chemical compounds that inhibited the enzyme's activity and severely affected the growth of this pathogen when tested in cultured cells.…”

Section: Introductionmentioning

confidence: 93%

“…The expression of histidine-tagged EhPLK in Escherichia coli and purification of the recombinant protein was done as described previously (Devi et al, 2019;Tarique et al, 2020). Briefly, transformed BL21(DE3) cells (Novagen) with EhPLK construct were cultured in Luria Bertani broth (Hi-Media) for overnight at 37°C and later secondary culture was inoculated with one percent of the overnight grown cells.…”

Section: Ehplk Protein Expression and Purificationmentioning

confidence: 99%

“…PLK kinetics was carried out as reported earlier (Tarique et al, 2020). Briefly, PLK enzymatic activity was determined in the absence of inhibitors for the reaction mixture containing EhPLK (25 mg/ml (0.40 mM), 2 mM MgCl 2 and 1 mM ATP, and activity was initiated with the addition of 20 mM of PL.…”

Section: In-vitro Inhibition Assaymentioning

confidence: 99%

“…We purchased five compounds on the basis of how well they docked in-silico onto the EhPLK structure (see docking scores in Table 1) at the PLP binding site (Figure 1) (Tarique et al, 2020) and their availability, to check their effects as inhibitors on the EhPLK enzyme. Amino acid residues at PLP binding site in EhPLK are Ser10, Val17, Leu41, His44, Thr45, Gly46, Tyr83, Tyr121 and Asp218.…”

Section: Ehplk Enzyme Kinetics In the Presence Of Inhibitorsmentioning

confidence: 99%

“…We have been extensively characterizing some of these enzymes as potential therapeutic targets for structure based drug discovery, (Kumar et al, 2011;Kumari et al, 2019;Singh et al, 2019;Dharavath et al, 2020) and what remained something of utmost importance was to target pyridoxal kinase itself, which catalyses the formation PLP in E. histolytica. In our previous work, we have biochemically characterized and determined the crystal structures of pyridoxal kinase from E. histolytica where a single copy of this gene exists for the synthesis of PLP through the salvage pathway (Tarique et al, 2020). Notable structural differences from human PLK in the active site region of this enzyme were observed which means targeting this enzyme would be detrimental and affect the activity of all PLP-dependent enzymes of this human pathogen.…”

Section: Introductionmentioning

confidence: 99%

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Inhibiting Pyridoxal Kinase of Entamoeba histolytica Is Lethal for This Pathogen

Devi

Tomar

Tarique

et al. 2021

Front. Cell. Infect. Microbiol.

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Pyridoxal 5’-phosphate (PLP) functions as a cofactor for hundreds of different enzymes that are crucial to the survival of microorganisms. PLP-dependent enzymes have been extensively characterized and proposed as drug targets in Entamoeba histolytica. This pathogen is unable to synthesize vitamin B6via de-novo pathway and relies on the uptake of vitamin B6 vitamers from the host which are then phosphorylated by the enzyme pyridoxal kinase to produce PLP, the active form of vitamin B6. Previous studies from our lab shows that EhPLK is essential for the survival and growth of this protozoan parasite and its active site differs significantly with respect to its human homologue making it a potential drug target. In-silico screening of EhPLK against small molecule libraries were performed and top five ranked molecules were shortlisted on the basis of docking scores. These compounds dock into the PLP binding site of the enzyme such that binding of these compounds hinders the binding of substrate. Of these five compounds, two compounds showed inhibitory activity with IC50 values between 100-250 μM when tested in-vitro. The effect of these compounds proved to be extremely lethal for Entamoeba trophozoites in cultured cells as the growth was hampered by 91.5% and 89.5% when grown in the presence of these compounds over the period of 72 hours.

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Section: Introductionmentioning

confidence: 93%

Section: Ehplk Protein Expression and Purificationmentioning

confidence: 99%

Section: In-vitro Inhibition Assaymentioning

confidence: 99%

Section: Ehplk Enzyme Kinetics In the Presence Of Inhibitorsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Inhibiting Pyridoxal Kinase of Entamoeba histolytica Is Lethal for This Pathogen

Devi

Tomar

Tarique

et al. 2021

Front. Cell. Infect. Microbiol.

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Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani

Roy,

Paul,

Lalchhuanawmi

et al. 2024

Biochimie

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Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate

De Vitto,

Belfon,

Sharma

et al. 2024

Biochemistry

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Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP.Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC 50 of 768 μM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

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Characterization and functional insights into the Entamoeba histolytica pyridoxal kinase, an enzyme essential for its survival

Cited by 6 publications

References 53 publications

Inhibiting Pyridoxal Kinase of Entamoeba histolytica Is Lethal for This Pathogen

Inhibiting Pyridoxal Kinase of Entamoeba histolytica Is Lethal for This Pathogen

Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani

Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate

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