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

Abstract

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.

Accelerated Reaction Engineering of Photo(bio)catalytic Reactions through Parallelization with an Open‐Source Photoreactor

“…The reactivity at different wavelengths was evaluated for the orthologue from Dinoroseobacter shibae DFL12T, revealing similar reaction rates at 460 nm (blue) and 641 nm (red) and a decreased rate at 542 nm (green) when using comparable radiant power [50] . Recently, we characterized several new members of the enzyme family [10] and decided to reinvestigate the wavelength dependence of the LPOR from Cyanobacterium aponinum using the open‐source photoreactor, screening four different wavelengths in parallel.…”

“…All reactions were performed as triplicates and using the four inner positions of a given row in the photoreactor with the outer positions occupied by dummy vials filled with water. The substrate protochlorophyllide ( 2 a ) was obtained from the Rhodobacter capsulatus ZY5 strain as described previously [10,63] …”

“…Until today only four light‐dependent groups of enzymes have been reported: [1] Flavin‐dependent photodecarboxylases were shown to decarboxylate medium to long‐chain fatty acids in a redox‐neutral process to the corresponding alkanes (Scheme 1, A) [8–9] . Protochlorophyllide oxidoreductases (LPORs) catalyze the stereo‐ and regio‐selective reduction of a C=C‐bond of excited protochlorophyllide (Scheme 1, B) [10–12] . CPD‐photolyases repair DNA by cleaving cyclobutane pyrimidine dimers (CPD) via a light dependent retro‐cycloaddition (not shown) [13–14] .…”

“…Herein we demonstrate how photobiocatalytic and photocatalytic reactions benefit from such a reactor by exemplifying accelerated reaction engineering of four photocatalytic reactions: (i) the fast identification of the ideal illumination conditions, reaction time and substrate concentration for the light dependent decarboxylation of palmitic acid 1 a using the photodecarboxylase from Chlorella variabilis ; [8] (ii) the straight‐forward identification of the ideal illumination wavelength for the protochlorophyllide oxidoreductase from Cyanobacterium aponinum ; [10] (iii) the evaluation of the inhibitory effect of amines on photosynthesis by Synechocystis sp . PCC 6083 and (iv) to complement the photobiocatalytic processes with a photoorganocatalytic example: the fast optimization and substrate screening of a (−)‐riboflavin‐catalyzed ( E / Z )‐isomerization of cinnamic acid derivatives as well as the application of this system to novel substrates [43] …”

Photo‐biocatalytic Cascades: Combining Chemical and Enzymatic Transformations Fueled by Light

“…[89] Several LPORs from different origins were biochemically characterized and applied in the light-driven conversion of pchlide to chlide under different conditions (light intensity, solvent content, pH range) and were analyzed for their cofactor flexibility. [90] Scheme 3. Schematic overview of strategies for the direct photoactivation of redox biocatalysts.…”