Mushroom tyrosinase oxidizes tyrosine-rich sequences to allow selective protein functionalization.
We show that mushroom tyrosinase catalyzes the formation of reactive o-quinones on unstructured, tyrosine-rich sequences such as hemagglutinin (HA) tags (YPYDVPDYA). In the absence of exogenous nucleophiles and at low protein concentrations, the o-quinone decomposes with fragmentation of the HA tag. At higher protein concentrations (>5 mg mL⁻¹), crosslinking is observed. Besthorn's reagent intercepts the o-quinone to give a characteristic pink complex that can be observed directly on a denaturing SDS-PAGE gel. Similar labeled species can be formed by using other nucleophiles such as Cy5-hydrazide. These reactions are selective for proteins bearing HA and other unstructured poly-tyrosine-containing tags and can be performed in lysates to create specifically tagged proteins.
Graduate Program in Biochemistry MS009, Brandeis University, 415 South Street, Waltham, MA 02453, USA.
Marcus J C
MJ
Long
Lizbeth
L
Hedstrom
D000363:Agaricales / Q000737:chemistry / Q000201:enzymology; D055162:Biocatalysis; D002232:Carbocyanines; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006388:Hemagglutinins / Q000737:chemistry*; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D011809:Quinones / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry
D002232:Carbocyanines; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D006388:Hemagglutinins; D010455:Peptides; D011809:Quinones; D011993:Recombinant Fusion Proteins; C085321:cyanine dye 5; C017663:polytyrosine; D014442:Monophenol Monooxygenase; D013762:Tetrahydrofolate Dehydrogenase