Enzyme Kinetics of 4-Hydroxyphenylacetate-1-hydroxylase
Advisor Information
John Conrad
Location
Dr. C.C. and Mabel L. Criss Library
Presentation Type
Poster
Start Date
7-3-2014 9:00 AM
End Date
7-3-2014 12:00 PM
Abstract
Flavin monooxygenases (FMOs) are a class of enzymes that use a tightly bound FAD prosthetic group and catalyze the incorporation of oxygen into an organic substrate. Typically bacterial FMOs are found in catabolic pathways breaking down organic substrates into metabolites that can be incorporated into energy pathways. This work focused on the relatively uncharacterized FMO 4-hydroxyphenylacetate-1-hydroxylase (4HPA1H), an FMO that catalyzes the hydroxylation of 4-hydroxyphenylatate (4HPA) forming 2,5-dihydroxyphenylacetate (HG or homogentistate). The gene for 4HPA1H from Delftia acidovorans was sub-cloned into the pET-14b plasmid, replicated in NEB-5α Competent E. Coli and the pET-14b-4HPA1H plasmid was used to transform BL-21 (DE3) competent E. Coli for the purpose of protein expression. Expression of 4HPA1H was induced in BL21s containing pET-14b-4HPA1H and protein was purified from inclusion bodies. Purification procedures have yielded purified and soluble protein.
Enzyme Kinetics of 4-Hydroxyphenylacetate-1-hydroxylase
Dr. C.C. and Mabel L. Criss Library
Flavin monooxygenases (FMOs) are a class of enzymes that use a tightly bound FAD prosthetic group and catalyze the incorporation of oxygen into an organic substrate. Typically bacterial FMOs are found in catabolic pathways breaking down organic substrates into metabolites that can be incorporated into energy pathways. This work focused on the relatively uncharacterized FMO 4-hydroxyphenylacetate-1-hydroxylase (4HPA1H), an FMO that catalyzes the hydroxylation of 4-hydroxyphenylatate (4HPA) forming 2,5-dihydroxyphenylacetate (HG or homogentistate). The gene for 4HPA1H from Delftia acidovorans was sub-cloned into the pET-14b plasmid, replicated in NEB-5α Competent E. Coli and the pET-14b-4HPA1H plasmid was used to transform BL-21 (DE3) competent E. Coli for the purpose of protein expression. Expression of 4HPA1H was induced in BL21s containing pET-14b-4HPA1H and protein was purified from inclusion bodies. Purification procedures have yielded purified and soluble protein.