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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 chains 1 – 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 present 5 – 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. A genetically encoded triplet photosensitizer is used to develop an efficient photoenzyme that can promote enantioselective intramolecular and bimolecular [2+2] cycloadditions by means of triplet energy transfer.

The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth 1 – 3 . POR, which is one of three known light-dependent enzymes 4 , 5 , catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide–NADPH–POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways. Crystal structures of cyanobacterial protochlorophyllide oxidoreductases reveal the basis of the photocatalytic activities of this enzyme, through the role of its active site in enabling the light-driven reduction of protochlorophyllide.

CarH is a coenzyme B 12 -dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B 12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B 12 -binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B 12 -ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B 12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes. CarH is a bacterial B 12 -binding photoreceptor involved in transcriptional regulation. Here, the authors provide insights into B 12 dynamics and associated cobalt redox changes following light activation. These demonstrate the CarH response integrates light and oxygen sensing.

We have used a combination of computational and structure-based redesign of the low molecular weight protein tyrosine phosphatase, LMW-PTP, to create new activity towards phosphoinositide substrates for which the wild-type enzyme had little or no activity. The redesigned enzymes retain catalytic activity despite residue alterations in the active site, and kinetic experiments confirmed specificity for up to four phosphoinositide substrates. Changes in the shape and overall volume of the active site where critical to facilitate access of the new substrates for catalysis. The kinetics data suggest that both the position and the combination of amino acid mutations are important for specificity towards the phosphoinositide substrates. The introduction of basic residues proved essential to establish new interactions with the multiple phosphate groups in the inositol head, thus promoting catalytically productive complexes. The crystallographic structures of the top-ranking designs confirmed the computational predictions and showed that residue substitutions do not alter the overall folding of the phosphatase or the conformation of the active site P-loop. The engineered LMW-PTP mutants with new activities can be useful reagents in investigating cell signalling pathways and offer the potential for therapeutic applications.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl- l -isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways 1 , 2 , 3 – 4 . Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown 1 , 2 . Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and l -Ile in plants 5 . This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions—Jar1 producing a compound essential for plant development 4 , 5 , and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications. A family of enzymes—coronafacic acid ligases, involved in the synthesis of bacterial phytotoxins—are found to catalyse amide bond formation with a wide range of substrates.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B 12 ) can act as a light-sensing chromophore heralded a new field of B 12 -photobiology. Although microbial genome analysis indicates that photoactive B 12 -binding domains form part of more complex protein architectures, regulating a range of molecular–cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B 12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B 12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B 12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B 12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts. Photoreceptor proteins utilise biological chromophores to regulate a large range of cellular processes in response to light. Here the authors identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that not only utilise B 12 but also contain biliverdin (BV) as an additional chromophore.

The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita–Baylis–Hillman (MBH) reaction. BH32.14 is suitable for preparative-scale transformations, accepts a broad range of aldehyde and enone coupling partners and is able to promote selective monofunctionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts (for example, thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not observed in nature. Directed evolution of a primitive computationally designed enzyme has produced an efficient and enantioselective biocatalyst for the Morita–Baylis–Hillman reaction. The engineered enzyme uses a designed histidine nucleophile operating in synergy with a catalytic arginine that emerged during evolution and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts.

The recent discovery of Is PETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable Is PETase variant (HotPETase, T m  = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes. Enzymes for poly(ethylene terephthalate) (PET) deconstruction are of interest for plastics recycling, but reports on their directed evolution are missing. Now, an automated, high-throughput directed evolution platform is described, affording HotPETase that effectively achieves depolymerization above the glass transition temperature of PET.

The rapid emergence of RNA therapeutics has highlighted the need for more efficient, scalable and sustainable methods for their manufacture. Biocatalytic approaches hold particular promise, but rely on a secure, sustainable and low-cost supply of nucleoside triphosphate (NTP) building blocks, including those containing chemical modifications. Here we report the development of a biocatalytic approach and engineered enzymes to convert widely available nucleosides into NTPs featuring pharmaceutically relevant modifications using inexpensive phosphate donors. Importantly our strategy obviates the need for ATP as a phosphate donor that complicates NTP isolation using existing methods. To showcase the utility of our approach, we employ an engineered acid phosphatase, polyphosphate kinase and acetate kinase to produce 2′- O -methoxyethyl-ATP (2′-MOE-ATP) and 2′-fluoro-ATP, key building blocks of commercial therapeutics. Finally, we show that crude NTPs from our process can be used directly in enzymatic oligonucleotide synthesis, obviating the need for costly NTP isolation or purification steps. Nucleoside triphosphates (NTPs) are important building blocks that underpin emerging enzymatic approaches to RNA therapeutics manufacturing. Here, authors develop a biocatalytic strategy to convert nucleosides into NTPs containing clinically relevant modifications, using simple phosphate donors.

Infection by soil transmitted parasitic helminths, such as Trichuris spp , are ubiquitous in humans and animals but the mechanisms determining persistence of chronic infections are poorly understood. Here we show that p43, the single most abundant protein in T. muris excretions/secretions, is non-immunogenic during infection and has an unusual sequence and structure containing subdomain homology to thrombospondin type 1 and interleukin (IL)−13 receptor (R) α2. Binding of p43 to IL-13, the key effector cytokine responsible for T. muris expulsion, inhibits IL-13 function both in vitro and in vivo. Tethering of p43 to matrix proteoglycans presents a bound source of p43 to facilitate interaction with IL-13, which may underpin chronic intestinal infection. Our results suggest that exploiting the biology of p43 may open up new approaches to modulating IL-13 function and control of Trichuris infections. In the study, the authors identify a protein excreted by the parasite Trichuris muris , p43, which can modulate IL-13 function, a key cytokine involved in host protection. These data suggest that p43 may be a novel therapeutic target for both whipworm infections and IL13 mediated pathologies.