Stability of recombinant yeast protoporphyrinogen oxidase: effects of diphenyl ether-type herbicides and diphenyleneiodonium.
Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.
Arnould
S
S
Laboratoire de Biochimie des Porphyrines, Département de Microbiologie, Institut Jacques-Monod, Paris, France.
Takahashi
M
M
Camadro
J M
JM
D001713:Biphenyl Compounds / Q000494:pharmacology; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006540:Herbicides / Q000494:pharmacology*; D007700:Kinetics; D009579:Nitrobenzoates / Q000494:pharmacology*; D009861:Onium Compounds / Q000494:pharmacology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011489:Protein Denaturation; D017510:Protein Folding; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae
D001713:Biphenyl Compounds; D006540:Herbicides; D009579:Nitrobenzoates; D009861:Onium Compounds; D011994:Recombinant Proteins; C007517:diphenyleneiodonium; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; C018425:acifluorfen