Enzymes of the last steps of chlorophyll biosynthesis: modification of the substrate structure helps to understand the topology of the active centers.
Enzymes catalyzing two of the late steps of chlorophyll biosynthesis are NADPH:protochlorophyllide oxidoreductase (POR), responsible for the light-dependent reduction of protochlorophyllide to chlorophyllide, and chlorophyll synthase that catalyses the esterification of chlorophyllide to chlorophyll. Inhibitors of these enzymes are of interest as potential herbicides. Both enzymes presumably form a complex, and the question arose whether chlorophyll synthase can react with chlorophyllide while it is still bound to POR. Here, we describe the chemical modification of protochlorophyllides and chlorophyllides with space-filling substituents at rings A, B, and E of the tetrapyrrole macrocycle and the reactivity of the modified substrates. Both enzymes tolerate the large and flexible phenylamino substituent at ring B, indicating that ring B points toward the enzyme surface while the substrate is bound. On the basis of the standard compound zinc protopheophorbide a (100% activity), the 7(1)-phenylamino derivative shows a comparable activity (83%) with POR that is higher than that of the parent formyl derivative zinc protopheophorbide b (58% activity). In contrast, the 3(1)-phenylamino derivative is less active (12%) than the parent formyl compound zinc protopheophorbide d (49% activity), indicating that the binding pocket leaves less space around ring A than around ring B. Almost no space must be left around ring E because substitution of the 13(2)-carboxymethyl ester (100% activity) by the 13(2)-carboxyethyl ester reduces the activity to 0.2%. Chlorophyll synthase leaves somewhat more space around ring E on the A side of the tetrapyrrole in the binding pocket; substitution of the 13(2)-proton (100% activity) by a methoxy group (53% activity) and an ethoxy group (11% activity) is tolerated to a certain extent, while the carbomethoxy group in this position is not accepted. Opening of ring E to a chlorin e6 dimethylester is tolerated (39% activity), while the large benzylamide residue at this site leads to the loss of activity. We conclude that the tetrapyrroles bind to both enzymes in the same direction: rings C, D, and E are oriented to the interior of the binding cleft, and rings A and B are oriented to the surface of the enzyme; this excludes simultaneous binding to both enzymes.
Rüdiger
Wolfhart
W
Department Biology I, Botany, University of München, Menzingerstrasse 67, D-80638 München, Germany. ruediger@lrz.uni-muenchen.de
Böhm
Stephan
S
Helfrich
Michael
M
Schulz
Stephanie
S
Schoch
Siegrid
S
D001426:Bacterial Proteins; D001665:Binding Sites; D019729:Carbon-Oxygen Ligases / Q000037:antagonists & inhibitors*; D002734:Chlorophyll / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000138:chemical synthesis; D006540:Herbicides; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D006540:Herbicides; D002734:Chlorophyll; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase; D019729:Carbon-Oxygen Ligases; C026901:chlorophyll synthetase