Overexpression and characterization of Escherichia coli dihydropyrimidine dehydrogenase: a four iron-sulphur cluster containing flavoprotein.
J Biochem
; 170(4): 511-520, 2021 Dec 04.
Article
in En
| MEDLINE
| ID: mdl-34097066
ABSTRACT
Escherichia coli dihydropyrimidine dehydrogenase (EcDPD) catalyses the NADH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines to control their metabolite pools. EcDPD consists of two subunits, PreT and PreA, and requires FAD, FMN and Fe-S clusters for activity. Recombinant EcDPD with a C-terminal His6-tagged-PreA subunit was overproduced in a DPD-lacking E. coli cells with augmented Fe-S cluster synthesis. Anaerobic purification resulted in purified enzyme with a specific activity of 13 µmol min-1 mg-1. The purified EcDPD was a heterotetramer and contained 0.81 FAD, 0.99 FMN, 14 acid-labile sulphur and 15 iron per PreT-PreA dimer. The enzyme exhibited Michaelis-Menten kinetics for both the forward and reverse reactions, which is distinct from mammalian DPDs showing substrate inhibition kinetics. For uracil reduction, the kcat, kcat/KNADH and kcat/Kuracil values were constant over the pH range of 5.5-10. For dihydrouracil (DHU) dehydrogenation, the pH-dependence of the kcat and kcat/KNAD+ values indicated that a residue with a pKa of 6.6 must be deprotonated for activity. Biochemical and kinetic comparisons with pig DPD revealed that protonation sates of the catalytically competent forms of EcDPD are distinct from those of pig enzyme.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Escherichia coli Proteins
/
Dihydrouracil Dehydrogenase (NADP)
/
Flavoproteins
/
Iron-Sulfur Proteins
Limits:
Animals
Language:
En
Journal:
J Biochem
Year:
2021
Document type:
Article
Affiliation country:
Japan