Photoreduction of Zinc Protopheophorbide b with NADPH-Protochlorophyllide Oxidoreductase from Etiolated Wheat (Triticum aestivum L.)

Lebedev

1999

Nature

117

letters to nature 80 NATURE | VOL 397 | 7 JANUARY 1999 | www.nature.com (Molecular Probes) was dissolved at 1 mg ml -1 in N-N-dimethylformamide and ionophoretically injected into the neuromuscular cleft. Embryos were placed in 1 3 PBS overnight at 4 8C, mounted in 50% glycerol plus 2% peraformaldehyde, and viewed on a Zeiss confocal microscope. Generation of transformants. Restriction fragments from lim3 genomic phage clones 9 were inserted into P[tau-myc] 4 or HZ50PL lacZ 28 plasmids and transformation was done as described 29 . Three independent lim3A-tau-myc lines and one lim3A-lacZ line were tested. All displayed similar expression patterns. Two of the lim3A-tau-myc insertions, denoted 1.8 and 1.13, were crossed into lim3 mutant backgrounds and gave identical results. UAS-lim3 was made by inserting the lim3 cDNA plus 50 base pairs of the Xenopus laevis b-globin 59 leader from plasmid pNB 25 into plasmid pUAS 18 . For the misexpression studies, several independent transgenic lines, each carrying one or two copies of UAS-lim3, were used. The ftz ng -GAL4.20 driver. To improve the expression levels of the original ftz ng -GAL4 line 19 , we generated 25 new insertion lines by transposase-mediated mobilization. Each line was tested for GAL4 expression by crossing it to¯ies carrying a UAS-tau-myc-GFP reporter 30 , where GFP is green¯uorescent protein. One line, ftz ng -GAL4.20, expressed GAL4 at high levels in most, if not all, motor neurons. As assayed by transactivation of the UAS-tau-myc reporter gene, 89% of hemisegments showed labelling in ISNb and 84% showed labelling in ISNd (n 56).

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confidence: 95%

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confidence: 99%

“…13) and ZnPPb (ref. 13), respectively (Pchlides a and b contain magnesium (Mg) instead of Zn; see ref. 13 for the structural formulas).…”

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confidence: 99%

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confidence: 99%

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confidence: 99%

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A protochlorophyllide light-harvesting complex involved in de-etiolation of higher plants

Lebedev

1999

Nature

117

Extending the Library of Light‐Dependent Protochlorophyllide Oxidoreductases and their Solvent Tolerance, Stability in Light and Cofactor Flexibility

et al. 2020

Biocatalysis is increasingly used in combination with light to develop new and more sustainable synthetic methods. Thereby, mostly a chemical photocatalyst harvesting the light energy is combined with an established enzymatic reaction, thus the biocatalyst itself does not require the light for its specific reaction. Here we expand the library of an enzyme which requires light for its natural reaction, namely the light‐dependent protochlorophyllide oxidoreductase (LPOR). This enzyme catalyzes the NADPH‐dependent reduction of a C=C in a N‐heterocycle. Out of five LPORs identified by sequence search, four were found to be well expressible in E. coli and active. Investigating the light intensity, which is an important parameter describing energy input and subsequently may enable fast reaction, it turned out that the four LPORs can stand the maximum light intensity reachable with the equipment used (1450 μmol photons m−2 s−1). However, the natural substrate and product were degraded at these conditions, allowing only 15 % of the maximum input (211 μmol photons m−2 s−1). Furthermore, the LPORs accepted seven different water miscible solvents with a solvent content of up to 20 % v/v and were active at a pH from 6 to 10. While all LPORs known to date are exclusively NADPH dependent, two LPORs identified here were active also with NADH. The cofactor selectivity could be pinned to three amino acid residues, which interestingly do not directly bind to the cofactor.

Protochlorophyllide b does not occur in barley etioplasts

et al. 1999

Self Cite

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection 6 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH:protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7 I -hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80^84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.z 1999 Federation of European Biochemical Societies.