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Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence
- Source :
- The Journal of Biological Chemistry, 295(47), 16013-16022. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, J Biol Chem, Journal of Biological Chemistry 295 (2020) 47, Journal of Biological Chemistry, 295(47), 16013-16022
- Publication Year :
- 2020
-
Abstract
- Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.
- Subjects :
- 0301 basic medicine
Luminescence
hydroxylase
Flavoprotein
Flavin group
Biochemistry
Cofactor
Mixed Function Oxygenases
Substrate Specificity
Fungal Proteins
03 medical and health sciences
chemistry.chemical_compound
Mycena chlorophos
enzyme kinetics
hispidin
Levensmiddelenchemie
Life Science
Enzyme kinetics
Molecular Biology
VLAG
chemistry.chemical_classification
Food Chemistry
030102 biochemistry & molecular biology
biology
protein engineering
Cell Biology
Monooxygenase
luciferase
flavin
Luciferin
bioluminescence
Recombinant Proteins
030104 developmental biology
Enzyme
chemistry
hispidin 3-hydroxylase
Enzymology
biology.protein
Hispidin
Agaricales
Subjects
Details
- Language :
- English
- ISSN :
- 00219258
- Database :
- OpenAIRE
- Journal :
- The Journal of Biological Chemistry, 295(47), 16013-16022. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, J Biol Chem, Journal of Biological Chemistry 295 (2020) 47, Journal of Biological Chemistry, 295(47), 16013-16022
- Accession number :
- edsair.doi.dedup.....b42915ad2c9ff76579956aeb7c8fa84e