Presentation Title

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.

This document is currently not available here.

COinS
 
Mar 7th, 9:00 AM Mar 7th, 12:00 PM

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.