The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Abstract

Protoporphyrinogen oxidase (EC 1-3-3-4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the betaalphabeta ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.

Figure 1

Proteolysis of protoporphyrinogen oxidase by trypsin. (A) Coomassie blue staining of proteins on an SDS-polyacrylamide gel. Lanes: 1, purified yeast protoporphyrinogen oxidase after 30-min incubation without trypsin; 2, purified yeast protoporphyrinogen oxidase after 30-min incubation with 25 units trypsin; 3, purified yeast protoporphyrinogen oxidase incubated with 10 μM acifluorfen after 30-min incubation with 25 units trypsin. (B) Protoporphyrinogen oxidase activity measured as a function of incubation time without trypsin (▴) and with 25 units trypsin (○).

Figure 2

Kinetics of purified protoporphyrinogen oxidase digestion by endoproteinase Glu-C. Lane C, enzyme incubated for 2 h without protease.

Figure 3

Fluorographic detection after SDS/PAGE analysis of purified yeast protoporphyrinogen oxidase in photoaffinity-labeling experiments with DZ-[³H]AF. (A) Intact enzyme. (B) Tryptic fragments of protoporphyrinogen oxidase. Lanes: M, molecular mass markers; −AF, total labeling; +AF, nonspecific labeling (labeling in the presence of 10 μM cold acifluorfen).

Figure 4

Fluorographic detection after SDS/PAGE of photolabeled tryptic fragments of the membrane-bound protoporphyrinogen oxidase fractionated by ultracentrifugation after the trypsin digestion. Lanes: 1 and 6, molecular mass markers; 2 and 4, total labeling in the membrane fraction (2) and supernatant (4); 3 and 5, nonspecific labeling (labeling in the presence of 10 μM cold acifluorfen) in the membrane fraction (3) and supernatant (5).

Figure 5

Effect of temperature on purified protoporphyrinogen oxidase (□) and the tryptic peptides (•) of protoporphyrinogen oxidase. The proteins were consecutively incubated for 5 min at each temperature, and the initial velocity of the enzyme reaction was measured at 30°C on aliquots of the denatured samples.

Figure 6

Organization of protoporphyrinogen oxidases from various origins and clustal-w 1.7 alignment of the peptide sequences of characteristic domains A, B, and J. ATh, Arabidopsis thaliana (34); NT, Nicotiana tabacum (35); HS, Homo sapiens (36); MM, Mus musculus (37); BS, Bacillus subtilis (30); MX, Myxococcus xanthus; SP, Schizosaccharomyces pombe; SC, Saccharomyces cerevisiae (9).