Active-Site Environmental Factors Customize the Photophysics of Photoenzymatic Old Yellow Enzymes.
ABSTRACT: The development of non-natural photoenzymatic systems has reinvigorated the study of photoinduced electron transfer (ET) within protein active sites, providing new and unique platforms for understanding how biological environments affect photochemical processes. In this work, we use ultrafast spectroscopy to compare the photoinduced electron transfer in known photoenzymes. 12-Oxophytodienoate reductase 1 (OPR1) is compared to Old Yellow Enzyme 1 (OYE1) and morphinone reductase (MR). The latter enzymes are structurally homologous to OPR1. We find that slight differences in the amino acid composition of the active sites of these proteins determine their distinct electron-transfer dynamics. Our work suggests that the inside of a protein active site is a complex/heterogeneous dielectric network where genetically programmed heterogeneity near the site of biological ET can significantly affect the presence and lifetime of various intermediate states. Our work motivates additional tunability of Old Yellow Enzyme active-site reorganization energy and electron-transfer energetics that could be leveraged for photoenzymatic redox approaches.
Project description:A photoenzymatic NADH regeneration system was established. The combination of deazariboflavin as a photocatalyst with putidaredoxin reductase enabled the selective reduction of NAD+ into the enzyme-active 1,4-NADH to promote an alcohol dehydrogenase catalysed stereospecific reduction reaction. The catalytic turnover of all the reaction components was demonstrated. Factors influencing the efficiency of the overall system were identified.
Project description:Flavin-dependent "ene"-reductases can generate stabilized alkyl radicals when irradiated with visible light; however, they are not known to form unstabilized radicals. Here, we report an enantioselective radical cyclization using alkyl iodides as precursors to unstabilized nucleophilic radicals. Evidence suggests this species is accessed by photoexcitation of a charge-transfer complex that forms between flavin and substrate within the protein active site. Stereoselective delivery of a hydrogen atom from the flavin semiquinone to the prochiral radical formed after cyclization provides high levels of enantioselectivity across a variety of substrates. Overall, this transformation demonstrates that photoenzymatic catalysis can address long-standing selectivity challenges in the radical literature.
Project description:Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop ?6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop ?6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for (S)-carvone reduction. Our findings demonstrate the contribution of loop ?6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.
Project description:Ene reductases from the Old Yellow Enzyme (OYE) family are industrially interesting enzymes for the biocatalytic asymmetric reduction of alkenes. To access both enantiomers of the target reduced products, stereocomplementary pairs of OYE enzymes are necessary, but their natural occurrence is quite limited. A library of wild type ene reductases from different sources was screened in the stereoselective reduction of a set of representative ?-alkyl-?-arylenones to investigate the naturally available biodiversity. As far as the bioreduction of the ethyl ketone derivatives concerns, the results confirmed the distinctiveness of the OYE3 enzyme in affording the reduced product in the (S) configuration, while all the other tested ene reductases from the Old Yellow Enzymes family showed the same stereoselectivity toward the formation of corresponding (R) enantiomer. A possible determinant role of the "hot spot" residue in position 296 for the stereoselectivity control of these reactions was confirmed by the replacement of Phe296 of OYE1 with Ser as found in OYE3. Further investigations showed that the same stereoselectivity switch in OYE1 could be achieved also by the replacement of Trp116 with Ala and Val, these experimental results being rationalized by structural and docking studies. Moreover, an additive effect on the stereoselectivity of OYE1 was observed when coupling the selected mutations in position 296 and 116, thus providing two extremely enantioselective variants of OYE1 (W116A-F296S, W116V-F296S) showing the opposite stereoselectivity of the wild type enzyme. Lastly, the effects of the mutations on the bioreduction of carvone enantiomers were investigated as well.
Project description:Controlled design of photoactive hybrids would provide highly active materials for solar energy conversion and photo(electro) catalysis. We describe the kinetics of photoinduced electron transfer in a series of photoactive hybrids based on Keggin-type polyoxometalates (POMs) covalently grafted to bodipy photosensitizers. We show how the electronic properties and corresponding dynamics of these hybrids can be readily tuned by varying the POM metal ion, the anchoring functionalization and the spacer length. Ultrafast visible and IR transient absorption spectroscopy, supported by spectroelectrochemical measurements, reveals that photoinduced electron transfer from the bodipy chromophore to the organosilyl POM derivative occurs as rapidly as <i>?</i> = 54 ps to generate a long-lived (<i>?</i> = 4.8 ns) charge-separated (CS) state, making this system appropriate for applications in photoelectrochemical devices.
Project description:Flavin-dependent ene-reductases (EREDs) are known to stereoselectively reduce activated alkenes, but are inactive toward carbonyls. Demonstrated here is that in the presence of photoredox catalysts, these enzymes will reduce aromatic ketones. Mechanistic experiments suggest this reaction proceeds through ketyl radical formation, a reaction pathway that is distinct from the native hydride-transfer mechanism. Furthermore, this reactivity is accessible without modification of either the enzyme or cofactors, allowing both native and non-natural mechanisms to occur simultaneously. Based on control experiments, we hypothesize that binding to the enzyme active site attenuates the reduction potential of the substrate, enabling single-electron reduction. This reactivity highlights opportunities to access new catalytic manifolds by merging photoredox catalysis with biocatalysis.
Project description:The reduction of C=C double bond, a key reaction in organic synthesis, is mostly achieved by traditional chemical methods. Therefore, the search for enzymes capable of performing this reaction is rapidly increasing. Old Yellow Enzymes (OYEs) are flavin-dependent oxidoreductases, initially isolated from Saccharomyces pastorianus. In this study, the presence and activation of putative OYE enzymes was investigated in the filamentous fungus Mucor circinelloides, which was previously found to mediate C=C reduction. Following an in silico approach, using S. pastorianus OYE1 amminoacidic sequence as template, ten putative genes were identified in the genome of M. circinelloides. A phylogenetic analysis revealed a high homology of McOYE1-9 with OYE1-like proteins while McOYE10 showed similarity with thermophilic-like OYEs. The activation of mcoyes was evaluated during the transformation of three different model substrates. Cyclohexenone, ?-methylcinnamaldehyde and methyl cinnamate were completely reduced in few hours and the induction of gene expression, assessed by qRT-PCR, was generally fast, suggesting a substrate-dependent activation. Eight genes were activated in the tested conditions suggesting that they may encode for active OYEs. Their expression over time correlated with C=C double bond reduction.
Project description:Threonine 37 is conserved among all the members of the old yellow enzyme (OYE) family. The hydroxyl group of this residue forms a hydrogen bond with the C-4 oxygen atom of the FMN reaction center of the enzyme [Fox, K. M. & Karplus, P. A. (1994) Structure 2, 1089-1105]. The position of Thr-37 and its interaction with flavin allow for speculations about its role in enzyme activity. This residue was mutated to alanine and the mutant enzyme was studied and compared with the wild-type OYE1 to evaluate its mechanistic function. The mutation has different effects on the two separate half-reactions of the enzyme. The mutant enzyme has enhanced activity in the oxidative half-reaction but the reductive half-reaction is slowed down by more than one order of magnitude. The peaks of the absorption spectra for enzyme bound with phenolic compounds are shifted toward shorter wavelengths than those of wild-type OYE1, consistent with its lower redox potential. It is suggested that Thr-37 in the wild-type OYE1 increases the redox potential of the enzyme by stabilizing the negative charge of the reduced flavin through hydrogen bonding with it.
Project description:Photoinduced electron transfer is central to many biological processes and technological applications, such as the harvesting of solar energy and molecular electronics. The electron donor and acceptor units involved in electron transfer are often held in place by covalent bonds, ?-? interactions or hydrogen bonds. Here, using time-resolved photoelectron spectroscopy and ab initio calculations, we reveal the existence of a new, low-energy, photoinduced electron-transfer mechanism in molecules held together by an NH?? bond. Specifically, we capture the electron-transfer process in a pyrrole dimer, from the excited ?-system of the donor pyrrole to a Rydberg orbital localized on the N-atom of the acceptor pyrrole, mediated by an N-H stretch on the acceptor molecule. The resulting charge-transfer state is surprisingly long lived and leads to efficient electronic relaxation. We propose that this relaxation pathway plays an important role in biological and technological systems containing the pyrrole building block.
Project description:Enoate reductases from the family of old yellow enzymes (OYEs) can catalyze stereoselective trans-hydrogenation of activated C=C bonds. Their application is limited by the necessity for a continuous supply of redox equivalents such as nicotinamide cofactors [NAD(P)H]. Visible light-driven activation of OYEs through NAD(P)H-free, direct transfer of photoexcited electrons from xanthene dyes to the prosthetic flavin moiety is reported. Spectroscopic and electrochemical analyses verified spontaneous association of rose bengal and its derivatives with OYEs. Illumination of a white light-emitting-diode triggered photoreduction of OYEs by xanthene dyes, which facilitated the enantioselective reduction of C=C bonds in the absence of NADH. The photoenzymatic conversion of 2-methylcyclohexenone resulted in enantiopure (ee>99?%) (R)-2-methylcyclohexanone with conversion yields as high as 80-90?%. The turnover frequency was significantly affected by the substitution of halogen atoms in xanthene dyes.