A robust high-throughput screening system to assess bacterial tyrosine ammonia lyase activity in the context of tyrosine inherited metabolic disorders.
High-throughput screening system
Hypertyrosinemia
Inborn errors of tyrosine metabolism
Tyrosine ammonia lyases
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
27 Sep 2024
27 Sep 2024
Historique:
received:
11
04
2024
accepted:
06
09
2024
medline:
28
9
2024
pubmed:
28
9
2024
entrez:
27
9
2024
Statut:
epublish
Résumé
Inborn errors of tyrosine metabolism result in patient's inability to degrade tyrosine. Current treatment consists of a phenylalanine and tyrosine restricted diet and nitisinone, causing a block in the tyrosine degradation pathway. However, tyrosine levels will increase, leading to acquired hypertyrosinemia, implying the need for an add-on treatment. Tyrosine ammonia lyases (TAL) can provide such an add-on treatment as they catalyze the deamination of tyrosine into p-coumaric acid and ammonia. In this study, we developed a robust high-throughput screening (HTS) assay to assess the capacity of bacterial TAL enzymes to decrease excessive tyrosine. The assay is based on the spectrophotometric quantification of p-coumaric acid after conversion of tyrosine by bacterial TAL. As a benchmark, TAL from Flavobacterium johnsoniae (FjTAL) was used to optimize the assay. Optimal growth conditions for high-level protein expression were determined by incubating transformed Escherichia coli BL21 (DE3) cells at different temperatures during various incubation times. Subsequently, assay temperature and pH were optimized followed by testing different ratios of tyrosine assay mixes to bacterial lysate. Finally, assay robustness and functionality were evaluated. Optimal FjTAL expression was obtained after incubation for 24 h at 22 °C. Ideal assay conditions consist of a 80/20 ratio of 1 mM tyrosine assay mix to FjTAL lysate performed at pH 9.2 and 37 °C. The robustness test showed Z' values > 0.4 and signal window values > 2 without edge or drift effects. As proof-of-principle, we successfully determined the catalytic activity of two other bacterial TAL enzymes RsTAL (5.718.10
Identifiants
pubmed: 39333684
doi: 10.1038/s41598-024-72360-9
pii: 10.1038/s41598-024-72360-9
doi:
Substances chimiques
Tyrosine
42HK56048U
Ammonia-Lyases
EC 4.3.1.-
L-tyrosine ammonia-lyase
EC 4.3.1.-
Bacterial Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
22175Subventions
Organisme : Fonds Wetenschappelijk Onderzoek
ID : G041521N
Organisme : Fonds Wetenschappelijk Onderzoek
ID : 11P1T24N
Organisme : Fonds Wetenschappelijk Onderzoek
ID : 11P3024N
Organisme : Fonds Wetenschappelijk Onderzoek
ID : G041521N
Organisme : Fonds Wetenschappelijk Onderzoek
ID : G041521N
Organisme : Fonds Wetenschappelijk Onderzoek
ID : G041521N
Informations de copyright
© 2024. The Author(s).
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