A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators
Summary: 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme involved in the pathogenesis of tyrosinemia III and cancer. Herein, we describe a spectroscopy-based assay to detect HPPD dioxygenase activity in the presence or absence of small-molecule modulators. We describe steps for transforma...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-06-01
|
| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723002678 |
| _version_ | 1797829491509166080 |
|---|---|
| author | Andrew Parkins Georgios Pantouris |
| author_facet | Andrew Parkins Georgios Pantouris |
| author_sort | Andrew Parkins |
| collection | DOAJ |
| description | Summary: 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme involved in the pathogenesis of tyrosinemia III and cancer. Herein, we describe a spectroscopy-based assay to detect HPPD dioxygenase activity in the presence or absence of small-molecule modulators. We describe steps for transformation, expression, and purification of HPPD and preparation of the assay plate. We detail initiation and completion of the enzymatic reaction followed by detection of remaining substrate in the form of enol-HPP/borate complex. This assay is applicable for high-throughput screening.For complete details on the use and execution of this protocol, please refer to Parkins et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-09T13:22:07Z |
| format | Article |
| id | doaj.art-7cc469add567430aafea553d5b8afba5 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-09T13:22:07Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-7cc469add567430aafea553d5b8afba52023-05-11T04:24:42ZengElsevierSTAR Protocols2666-16672023-06-0142102300A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulatorsAndrew Parkins0Georgios Pantouris1University of the Pacific, Stockton, CA 95211, USAUniversity of the Pacific, Stockton, CA 95211, USA; Corresponding authorSummary: 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme involved in the pathogenesis of tyrosinemia III and cancer. Herein, we describe a spectroscopy-based assay to detect HPPD dioxygenase activity in the presence or absence of small-molecule modulators. We describe steps for transformation, expression, and purification of HPPD and preparation of the assay plate. We detail initiation and completion of the enzymatic reaction followed by detection of remaining substrate in the form of enol-HPP/borate complex. This assay is applicable for high-throughput screening.For complete details on the use and execution of this protocol, please refer to Parkins et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723002678CancerHealth SciencesHigh throughput screeningProtein expression and purification |
| spellingShingle | Andrew Parkins Georgios Pantouris A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators STAR Protocols Cancer Health Sciences High throughput screening Protein expression and purification |
| title | A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators |
| title_full | A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators |
| title_fullStr | A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators |
| title_full_unstemmed | A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators |
| title_short | A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators |
| title_sort | novel enzymatic assay for the identification of 4 hydroxyphenylpyruvate dioxygenase modulators |
| topic | Cancer Health Sciences High throughput screening Protein expression and purification |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723002678 |
| work_keys_str_mv | AT andrewparkins anovelenzymaticassayfortheidentificationof4hydroxyphenylpyruvatedioxygenasemodulators AT georgiospantouris anovelenzymaticassayfortheidentificationof4hydroxyphenylpyruvatedioxygenasemodulators AT andrewparkins novelenzymaticassayfortheidentificationof4hydroxyphenylpyruvatedioxygenasemodulators AT georgiospantouris novelenzymaticassayfortheidentificationof4hydroxyphenylpyruvatedioxygenasemodulators |