Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

A light-driven enzymatic enantioselective radical acylation.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1-3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6-8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools.

Enantioselective [2+2]-cycloadditions with triplet photoenzymes.

Naturally evolved enzymes, despite their astonishingly large variety and functional diversity, operate predominantly through thermochemical activation. Integrating prominent photocatalysis modes into proteins, such as triplet energy transfer, could create artificial photoenzymes that expand the scope of natural biocatalysis1-3. Here, we exploit genetically reprogrammed, chemically evolved photoenzymes embedded with a synthetic triplet photosensitizer that are capable of excited-state enantio-induction4-6. Structural optimization through four rounds of directed evolution afforded proficient variants for the enantioselective intramolecular [2+2]-photocycloaddition of indole derivatives with good substrate generality and excellent enantioselectivities (up to 99% enantiomeric excess). A crystal structure of the photoenzyme-substrate complex elucidated the non-covalent interactions that mediate the reaction stereochemistry. This study expands the energy transfer reactivity7-10 of artificial triplet photoenzymes in a supramolecular protein cavity and unlocks an integrated approach to valuable enantioselective photochemical synthesis that is not accessible with either the synthetic or the biological world alone.

A designed photoenzyme for enantioselective [2+2] cycloadditions.

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Quaternary Charge-Transfer Complex Enables Photoenzymatic Intermolecular Hydroalkylation of Olefins.

Intermolecular C-C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.

Photoenzymatic enantioselective intermolecular radical hydroalkylation.

Enzymes are increasingly explored for use in asymmetric synthesis1-3, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis4 has spurred the discovery of novel reactivity from known enzymes5. However, so far photoinduced enzymatic catalysis6 has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate7. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an 'ene' reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 per cent yield with 99 per cent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the 'ene' reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.

Coupling natural systems with synthetic chemistry for light-driven enzymatic biocatalysis.

Visible light-driven redox reactions have been widely adopted for the production of chemicals to combat energy shortage and global warming. Key elements of such a reaction system include a photosensitizer, a catalyst, and an electron source. In this review, we introduce the small molecules and nanoparticles that are widely used as photosensitizers, as well as the development of a photosensitizer protein that is based on the expansion of genetic code, with a fluorescent protein that is used as a scaffold. Visible light-driven enzymes using proteins as photosensitizers or as catalysts such as carbon monoxide dehydrogenase (CODH), formic acid dehydrogenase (FDH), hydrogenase, nitrogenase, cytochrome P450 BM3, and alkane synthase are then described. CODH can be coupled with photosensitizing nanoparticles to reduce CO2 to CO, and hydrogenase can produce H2 using high-energy electrons produced from dye-sensitized nanoparticles. When water-soluble zinc porphyrin is coupled with FDH, visible light drives CO2 to produce formic acid. Nitrogenase can reduce N2 to NH3 using CdS nanoparticle as photosensitizer. Cytochrome P450 BM3 can be enhanced by a visible light-driven redox system and thus by hydroxylate lauric acid or fatty acids. CvFAP, an alkane synthase, can decarboxylate palmitic acid to pentadecane under blue light excitation. Moreover, we describe a genetically encoded photosensitive protein, which mimics the function of natural photosynthesis and catalyzes the conversion of CO2 to CO when covalently attached with a Ni-terpyridine complex.

Influence of UV radiation on molecular structure and catalytic activity of free and immobilized bromelain, ficin and papain.

Our research has shown that the degree of photosensitivity of the cysteine proteases can be arranged in the following order: bromelain → ficin → papain. After the UV irradiation with 151 J·m-2 intensity of a bromelain solution, the enzyme activity has increased. No decrease in the catalytic capacity and the change in the size of the molecule was recorded in the 151-6040 J·m-2 range of irradiation intensities. A decrease in the catalytic capacity of ficin and the increase of its globule size occurred after exposure to a radiation of 3020 J·m-2 intensity. The decrease in papain activity was observed at the UV irradiation intensity of 453 J·m-2, and an increase of the papain globule size was detected at 755 J·m-2. Immobilization on chitosan matrix leads to the increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be postulated that the chitosan matrix acts as photoprotector for immobilized ficin, bromelain and papain. The obtained results can be helpful for development of drugs based on chitosan and cysteine proteases in combination with phototherapy, as well as for choosing their sterilization conditions.

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology.

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of ~100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex.

Coupling efficiency in light-driven hybrid P450BM3 and CYP119 enzymes.

The light-driven hybrid P450 enzyme approach utilizing the photochemical properties of a covalently attached Ru(II)-diimine photosensitizer was extended to the archaeal Sulfolobus acidocaldarius CYP119 enzyme leading to high photocatalytic activity in the hydroxylation of the chromogenic substrate, 11-nitrophenoxyundecanoic acid. The determined kcat was greater than those reported with various natural redox partners. In addition, the sacrificial electron donor, diethyldithiocarbamate, used in the photocatalytic reaction is shown to play a dual role. It acts as an efficient quencher of the Ru(II) excited state leading to a highly reducing species necessary to inject electrons into the heme. It is also known for its antioxidant properties and is shown herein to be a useful probe to determine coupling efficiency in the light-driven hybrid enzymes.

Photoexcitation of flavoenzymes enables a stereoselective radical cyclization.

Photoexcitation is a common strategy for initiating radical reactions in chemical synthesis. We found that photoexcitation of flavin-dependent "ene"-reductases changes their catalytic function, enabling these enzymes to promote an asymmetric radical cyclization. This reactivity enables the construction of five-, six-, seven-, and eight-membered lactams with stereochemical preference conferred by the enzyme active site. After formation of a prochiral radical, the enzyme guides the delivery of a hydrogen atom from flavin-a challenging feat for small-molecule chemical reagents. The initial electron transfer occurs through direct excitation of an electron donor-acceptor complex that forms between the substrate and the reduced flavin cofactor within the enzyme active site. Photoexcitation of promiscuous flavoenzymes has thus furnished a previously unknown biocatalytic reaction.

Photoreduction of CO2 with a Formate Dehydrogenase Driven by Photosystem II Using a Semi-artificial Z-Scheme Architecture.

Solar-driven coupling of water oxidation with CO2 reduction sustains life on our planet and is of high priority in contemporary energy research. Here, we report a photoelectrochemical tandem device that performs photocatalytic reduction of CO2 to formate. We employ a semi-artificial design, which wires a W-dependent formate dehydrogenase (FDH) cathode to a photoanode containing the photosynthetic water oxidation enzyme, Photosystem II, via a synthetic dye with complementary light absorption. From a biological perspective, the system achieves a metabolically inaccessible pathway of light-driven CO2 fixation to formate. From a synthetic point of view, it represents a proof-of-principle system utilizing precious-metal-free catalysts for selective CO2-to-formate conversion using water as an electron donor. This hybrid platform demonstrates the translatability and versatility of coupling abiotic and biotic components to create challenging models for solar fuel and chemical synthesis.

Cooperative asymmetric reactions combining photocatalysis and enzymatic catalysis.

Living organisms rely on simultaneous reactions catalysed by mutually compatible and selective enzymes to synthesize complex natural products and other metabolites. To combine the advantages of these biological systems with the reactivity of artificial chemical catalysts, chemists have devised sequential, concurrent, and cooperative chemoenzymatic reactions that combine enzymatic and artificial catalysts1-9. Cooperative chemoenzymatic reactions consist of interconnected processes that generate products in yields and selectivities that cannot be obtained when the two reactions are carried out sequentially with their respective substrates2,7. However, such reactions are difficult to develop because chemical and enzymatic catalysts generally operate in different media at different temperatures and can deactivate each other1-9. Owing to these constraints, the vast majority of cooperative chemoenzymatic processes that have been reported over the past 30 years can be divided into just two categories: chemoenzymatic dynamic kinetic resolutions of racemic alcohols and amines, and enzymatic reactions requiring the simultaneous regeneration of a cofactor2,4,5. New approaches to the development of chemoenzymatic reactions are needed to enable valuable chemical transformations beyond this scope. Here we report a class of cooperative chemoenzymatic reaction that combines photocatalysts that isomerize alkenes with ene-reductases that reduce carbon-carbon double bonds to generate valuable enantioenriched products. This method enables the stereoconvergent reduction of E/Z mixtures of alkenes or reduction of the unreactive stereoisomers of alkenes in yields and enantiomeric excesses that match those obtained from the reduction of the pure, more reactive isomers. The system affords a range of enantioenriched precursors to biologically active compounds. More generally, these results show that the compatibility between photocatalysts and enzymes enables chemoenzymatic processes beyond cofactor regeneration and provides a general strategy for converting stereoselective enzymatic reactions into stereoconvergent ones.

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water by Steccherinum murashkinskyi laccase: insights into the reaction mechanism.

The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35 Šresolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.

Light-sensing via hydrogen peroxide and a peroxiredoxin.

Yeast lacks dedicated photoreceptors; however, blue light still causes pronounced oscillations of the transcription factor Msn2 into and out of the nucleus. Here we show that this poorly understood phenomenon is initiated by a peroxisomal oxidase, which converts light into a hydrogen peroxide (H2O2) signal that is sensed by the peroxiredoxin Tsa1 and transduced to thioredoxin, to counteract PKA-dependent Msn2 phosphorylation. Upon H2O2, the nuclear retention of PKA catalytic subunits, which contributes to delayed Msn2 nuclear concentration, is antagonized in a Tsa1-dependent manner. Conversely, peroxiredoxin hyperoxidation interrupts the H2O2 signal and drives Msn2 oscillations by superimposing on PKA feedback regulation. Our data identify a mechanism by which light could be sensed in all cells lacking dedicated photoreceptors. In particular, the use of H2O2 as a second messenger in signalling is common to Msn2 oscillations and to light-induced entrainment of circadian rhythms and suggests conserved roles for peroxiredoxins in endogenous rhythms.

RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique µ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.

Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light.

Enzymes are ideal for use in asymmetric catalysis by the chemical industry, because their chemical compositions can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biologically mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide co-factors (molecules that assist enzyme-mediated catalysis). Under irradiation with visible light, the nicotinamide-dependent enzyme known as ketoreductase can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones-a challenging transformation for small-molecule catalysts. Mechanistic experiments support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and nicotinamide adenine dinucleotide phosphate, respectively) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photo-induced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biological co-factors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of co-factor regeneration.

Rhodobacter sphaeroides CryB is a bacterial cryptochrome with (6-4) photolyase activity.

Photolyases are efficient DNA repair enzymes that specifically repair either cyclobutane pyrimidine dimers or (6-4) photoproducts in a light-dependent cleavage reaction. The closely related classical cryptochrome blue light photoreceptors do not repair DNA lesions; instead they are involved in regulatory processes. CryB of Rhodobacter sphaeroides was until now described as a cryptochrome that affects light-dependent and singlet oxygen-dependent gene expression and is unusual in terms of its cofactor composition. Here we present evidence for a repair activity of (6-4) photoproducts by CryB and suggest a dual character combining the functions of cryptochromes and photolyases. We investigated the effects of crucial amino acids involved in cofactor or DNA lesion binding on the light-dependent recovery of cells after UV light exposure (in vivo photoreactivation). Remarkably, impairment of one of the two light absorbing cofactors, FAD or 6,7-dimethyl-8-ribityllumazine, only marginally affected the final survival rate but strongly decelerated photoreactivation kinetics. The impairment of both of them together through mutagenesis decreased CryB-dependent photoreactivation to the level of the ∆cryB knockout strain. The third cofactor, a [4Fe4S] iron-sulfur cluster, is indispensable for the structural integrity of the protein. The reduction of FAD via the conserved tryptophan W338, which is crucial for in vitro reduction and consequently DNA repair, is not required for in vivo photoreactivation, suggesting that this reduction pathway to FAD is dispensable in the cellular environment. This demonstrates that in vitro experiments give only limited information on in vivo photolyase activity.

Biosynthesis of titanium dioxide nanoparticles using Bacillus amyloliquefaciens culture and enhancement of its photocatalytic activity for the degradation of a sulfonated textile dye Reactive Red 31.

The present study aims at exploiting Bacillus amyloliquefaciens for the biosynthesis of titanium dioxide nanoparticles and also investigates role of bacterial enzymes in the biosynthesis of titanium dioxide nanoparticles. Bacterial synthesized as well as metal doped titanium dioxide nanoparticles were characterized by X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), Energy dispersive X-ray spectroscopy (EDAX). Amylase activity (43.37IU) in culture supernatant evinced a potential involvement of extracellular enzyme in TiO2 nanoparticle biosynthesis. Crystallite size of bio-synthesized nanoparticles was found to be in the range of 15.23-87.6nm. FTIR spectroscopy and native-PAGE (Polyacrylamide Gel Electrophoresis) clearly indicated involvement of alpha amylase in biosynthesis of TiO2 nanoparticles and in their stabilization. TEM micrographs of the synthesized titanium dioxide nanoparticles revealed the formation of spherical nanoparticles with a size range of 22.11-97.28nm. Photocatalytic degradation of Reactive Red 31 (RR31) dye was carried out using bio-synthesized TiO2 nanoparticles under UV radiation. Photocatalytic activity of synthesized nanoparticles was enhanced by Ag, La, Zn and Pt doping. Platinum doped TiO2 showed highest potential (90.98%) in RR31 degradation as compared to undoped (75.83%).