An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1

Andrew G. Wagner; Roozbeh Eskandari; Vern L. Schramm

doi:10.1016/j.ab.2023.115171

An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1

Andrew G. Wagner, Roozbeh Eskandari, Vern L. Schramm

Biochemistry

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

2ʹ-Deoxynucleoside 5ʹ-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2ʹ-deoxyuridine 5ʹ-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.

Original language	English (US)
Article number	115171
Journal	Analytical Biochemistry
Volume	672
DOIs	https://doi.org/10.1016/j.ab.2023.115171
State	Published - Jul 1 2023

Keywords

2-Deoxyribose 5-phosphate aldolase
5-Hydroxymethyl uracil
DNPH1
Epigenetics
N-deoxynucleotide glycosidase
Pyrimidine salvage

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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title = "An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1",

abstract = "2ʹ-Deoxynucleoside 5ʹ-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2ʹ-deoxyuridine 5ʹ-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.",

keywords = "2-Deoxyribose 5-phosphate aldolase, 5-Hydroxymethyl uracil, DNPH1, Epigenetics, N-deoxynucleotide glycosidase, Pyrimidine salvage",

author = "Wagner, {Andrew G.} and Roozbeh Eskandari and Schramm, {Vern L.}",

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doi = "10.1016/j.ab.2023.115171",

language = "English (US)",

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T1 - An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1

AU - Wagner, Andrew G.

AU - Eskandari, Roozbeh

AU - Schramm, Vern L.

PY - 2023/7/1

Y1 - 2023/7/1

N2 - 2ʹ-Deoxynucleoside 5ʹ-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2ʹ-deoxyuridine 5ʹ-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.

AB - 2ʹ-Deoxynucleoside 5ʹ-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2ʹ-deoxyuridine 5ʹ-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.

KW - 2-Deoxyribose 5-phosphate aldolase

KW - 5-Hydroxymethyl uracil

KW - DNPH1

KW - Epigenetics

KW - N-deoxynucleotide glycosidase

KW - Pyrimidine salvage

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JO - Analytical Biochemistry

JF - Analytical Biochemistry

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An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1

Abstract

Keywords

ASJC Scopus subject areas

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