Current Understanding of the Mechanism of Water Oxidation in Photosystem II and Its Relation to XFEL Data.

The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.

Role of Mammalian DNA Methyltransferases in Development.

DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.

Moving Through Barriers in Science and Life.

This first serious attempt at an autobiographical accounting has forced me to sit still long enough to compile my thoughts about a long personal and scientific journey. I especially hope that my trajectory will be of interest and perhaps beneficial to much younger women who are just getting started in their careers. To paraphrase from Virginia Woolf's writings in A Room of One's Own at the beginning of the 20th century, "for most of history Anonymous was a Woman." However, Ms. Woolf is also quoted as saying "nothing has really happened until it has been described," a harbinger of the enormous historical changes that were about to be enacted and recorded by women in the sciences and other disciplines. The progress in my chosen field of study-the chemical basis of enzyme action-has also been remarkable, from the first description of an enzyme's 3D structure to a growing and deep understanding of the origins of enzyme catalysis.

A Rich Man, Poor Man Story of S-Adenosylmethionine and Cobalamin Revisited.

S-adenosylmethionine (AdoMet) has been referred to as both "a poor man's adenosylcobalamin (AdoCbl)" and "a rich man's AdoCbl," but today, with the ever-increasing number of functions attributed to each cofactor, both appear equally rich and surprising. The recent characterization of an organometallic species in an AdoMet radical enzyme suggests that the line that differentiates them in nature will be constantly challenged. Here, we compare and contrast AdoMet and cobalamin (Cbl) and consider why Cbl-dependent AdoMet radical enzymes require two cofactors that are so similar in their reactivity. We further carry out structural comparisons employing the recently determined crystal structure of oxetanocin-A biosynthetic enzyme OxsB, the first three-dimensional structural data on a Cbl-dependent AdoMet radical enzyme. We find that the structural motifs responsible for housing the AdoMet radical machinery are largely conserved, whereas the motifs responsible for binding additional cofactors are much more varied.

Biochemistry of Catabolic Reductive Dehalogenation.

A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.

Biosynthesis of the Metalloclusters of Nitrogenases.

Nitrogenase is a versatile metalloenzyme that is capable of catalyzing two important reactions under ambient conditions: the reduction of nitrogen (N2) to ammonia (NH3), a key step in the global nitrogen cycle; and the reduction of carbon monoxide (CO) and carbon dioxide (CO2) to hydrocarbons, two reactions useful for recycling carbon waste into carbon fuel. The molybdenum (Mo)- and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Each of them contains a P-cluster and a cofactor, two high-nuclearity metalloclusters that have crucial roles in catalysis. This review summarizes the progress that has been made in elucidating the biosynthetic mechanisms of the P-cluster and cofactor species of nitrogenase, focusing on what is known about the assembly mechanisms of the two metalloclusters in Mo-nitrogenase and giving a brief account of the possible assembly schemes of their counterparts in V-nitrogenase, which are derived from the homology between the two nitrogenases.

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Emerging paradigms for complex iron-sulfur cofactor assembly and insertion.

[FeFe]-hydrogenses and molybdenum (Mo)-nitrogenase are evolutionarily unrelated enzymes with unique complex iron-sulfur cofactors at their active sites. The H cluster of [FeFe]-hydrogenases and the FeMo cofactor of Mo-nitrogenase require specific maturation machinery for their proper synthesis and insertion into the structural enzymes. Recent insights reveal striking similarities in the biosynthetic pathways of these complex cofactors. For both systems, simple iron-sulfur cluster precursors are modified on assembly scaffolds by the activity of radical S-adenosylmethionine (SAM) enzymes. Radical SAM enzymes are responsible for the synthesis and insertion of the unique nonprotein ligands presumed to be key structural determinants for their respective catalytic activities. Maturation culminates in the transfer of the intact cluster assemblies to a cofactor-less structural protein recipient. Required roles for nucleotide binding and hydrolysis have been implicated in both systems, but the specific role for these requirements remain unclear. In this review, we highlight the progress on [FeFe]-hydrogenase H cluster and nitrogenase FeMo-cofactor assembly in the context of these emerging paradigms.

Class I ribonucleotide reductases: metallocofactor assembly and repair in vitro and in vivo.

Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an Fe(III)Fe(III)-tyrosyl radical (Y•) cofactor (class Ia), a Mn(III)Mn(III)-Y• cofactor (class Ib), and a Mn(IV)Fe(III) cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our understanding of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields.

Structure of the complex I-like molecule NDH of oxygenic photosynthesis.

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.

Enzymatic assembly of carbon-carbon bonds via iron-catalysed sp3 C-H functionalization.

Although abundant in organic molecules, carbon-hydrogen (C-H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C-H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp3 carbon1,2. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp3 C-H bonds through carbene C-H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C-H functionalization1,2. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C-H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp3 C-H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C-H functionalization reactions for applications in chemistry and synthetic biology.

A complete biomimetic iron-sulfur cubane redox series.

Synthetic iron-sulfur cubanes are models for biological cofactors, which are essential to delineate oxidation states in the more complex enzymatic systems. However, a complete series of [Fe4S4]n complexes spanning all redox states accessible by 1-electron transformations of the individual iron atoms (n = 0-4+) has never been prepared, deterring the methodical comparison of structure and spectroscopic signature. Here, we demonstrate that the use of a bulky arylthiolate ligand promoting the encapsulation of alkali-metal cations in the vicinity of the cubane enables the synthesis of such a series. Characterization by EPR, 57Fe Mössbauer spectroscopy, UV-visible electronic absorption, variable-temperature X-ray diffraction analysis, and cyclic voltammetry reveals key trends for the geometry of the Fe4S4 core as well as for the Mössbauer isomer shift, which both correlate systematically with oxidation state. Furthermore, we confirm the S = 4 electronic ground state of the most reduced member of the series, [Fe4S4]0, and provide electrochemical evidence that it is accessible within 0.82 V from the [Fe4S4]2+ state, highlighting its relevance as a mimic of the nitrogenase iron protein cluster.

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT.

The maturation of RAS GTPases and approximately 200 other cellular CAAX proteins involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CAAX motif, proteolytic cleavage of the AAX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate. This final step is catalysed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryote-specific integral membrane enzyme that resides in the endoplasmic reticulum. ICMT is the only cellular enzyme that is known to methylate prenylcysteine substrates; methylation is important for the biological functions of these substrates, such as the membrane localization and subsequent activity of RAS, prelamin A and RAB. Inhibition of ICMT has potential for combating progeria and cancer. Here we present an X-ray structure of ICMT, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor, at 2.3 Å resolution. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for the cytosolic methyl donor, S-adenosyl-l-methionine, and for prenylcysteine substrates, which are associated with the endoplasmic reticulum membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localizations together in a membrane environment-a relatively uncharacterized but defining feature of many integral membrane enzymes.

Long-range structural defects by pathogenic mutations in most severe glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common blood disorder, presenting multiple symptoms, including hemolytic anemia. It affects 400 million people worldwide, with more than 160 single mutations reported in G6PD. The most severe mutations (about 70) are classified as class I, leading to more than 90% loss of activity of the wild-type G6PD. The crystal structure of G6PD reveals these mutations are located away from the active site, concentrating around the noncatalytic NADP+-binding site and the dimer interface. However, the molecular mechanisms of class I mutant dysfunction have remained elusive, hindering the development of efficient therapies. To resolve this, we performed integral structural characterization of five G6PD mutants, including four class I mutants, associated with the noncatalytic NADP+ and dimerization, using crystallography, small-angle X-ray scattering (SAXS), cryogenic electron microscopy (cryo-EM), and biophysical analyses. Comparisons with the structure and properties of the wild-type enzyme, together with molecular dynamics simulations, bring forward a universal mechanism for this severe G6PD deficiency due to the class I mutations. We highlight the role of the noncatalytic NADP+-binding site that is crucial for stabilization and ordering two ß-strands in the dimer interface, which together communicate these distant structural aberrations to the active site through a network of additional interactions. This understanding elucidates potential paths for drug development targeting G6PD deficiency.

Acetogenic bacteria utilize light-driven electrons as an energy source for autotrophic growth.

Acetogenic bacteria use cellular redox energy to convert CO2 to acetate using the Wood-Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as from inorganic materials, such as photoresponsive semiconductors. We have developed a nanoparticle-microbe hybrid system in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) are displayed on the cell surface of the industrial acetogen Clostridium autoethanogenum The hybrid system converts CO2 into acetate without the need for additional energy sources, such as H2, and uses only light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum uses electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicate that genes encoding the metal ion or flavin-binding proteins were highly up-regulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

A second riboswitch class for the enzyme cofactor NAD.

A bacterial noncoding RNA motif almost exclusively associated with pnuC genes was uncovered using comparative sequence analysis. Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD+). Thus, we speculated that the newly found "pnuC motif" RNAs might function as aptamers for a novel class of NAD+-sensing riboswitches. RNA constructs that encompass the conserved nucleotides and secondary structure features that define the motif indeed selectively bind NAD+, nicotinamide mononucleotide (NMN), and NR. Mutations that disrupt strictly conserved nucleotides of the aptamer also disrupt ligand binding. These bioinformatic and biochemical findings indicate that pnuC motif RNAs are likely members of a second riboswitch class that regulates gene expression in response to NAD+ binding.

Photobiocatalysis for Abiological Transformations.

Harnessing biocatalysts for novel abiological transformations is a longstanding goal of synthetic chemistry. Combining the merits of biocatalysis and photocatalysis allows for selective transformations fueled by visible light and offers many advantages including new reactivity, high enantioselectivity, greener syntheses, and high yields. Photoinduced electron or energy transfer enables synthetic methodologies that complement conventional two electron processes or offer orthogonal pathways for developing new reactions. Enzymes are well suited and can be tuned by directed evolution to exert control over open-shell intermediates, thereby suppressing undesirable reactions and delivering high chemo- and stereoselectivities. Within the past decade, the combination of biocatalysis and photocatalysis was mainly focused on exploiting light-regenerated cofactors to function native enzymatic activity. However, recent developments have demonstrated that the combination can unlock new-to-nature chemistry. Particularly, the discovery and application of new strategies are well poised to expand the applications of photobiocatalysis.In the past five years, our lab has been studying the combinations of photocatalysis and biocatalysis that can be applied to create new synthetic methodologies and solve challenges in synthetic organic chemistry. Our efforts have expanded the strategies for combining external photocatalysts with enzymes through the construction of a synergistic cooperative stereoconvergent reduction system consisting of photosensitized energy transfer and ene-reductase-catalyzed alkene reduction. Additionally, our efforts have also extended the capability of cofactor-dependent photoenzymatic systems to include enantioselective bimolecular radical hydroalkylations of alkenes by irradiating electron donor-acceptor complexes comprised of enzymatic redox active cofactors and unnatural substrates.In this Account, we highlight strategies developed by our group and others for combining biocatalysis and photocatalysis with the aim of introducing non-natural reactivity to enzymes. Presently, strategies applied to achieve this goal include the repurposing of natural photoenzymes, the elucidation of new photoreactivity within cofactor-dependent enzymes, the combination of external photocatalysts with enzymes, and the construction of artificial photoenzymes. By demonstrating the successful applications of these strategies for achieving selective new-to-nature transformations, we hope to spur interest in expanding the scope of photobiocatalytic systems through the use and extension of these strategies and creation of new strategies. Additionally, we hope to elucidate the intuition in synergizing the unique capabilities of biocatalysis and photocatalysis so that photobiocatalysis can be recognized as a potential solution to difficult challenges in synthetic organic chemistry.

Design of thiamine analogues for inhibition of thiamine diphosphate (ThDP)-dependent enzymes: Systematic investigation through Scaffold-Hopping and C2-Functionalisation.

Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positively charged thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) which is the best aromatic ring? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can further improve both potency and selectivity.

Elucidation of the biosynthesis of the methane catalyst coenzyme F430.

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA-CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.

DNAmoreDB, a database of DNAzymes.

Deoxyribozymes, DNA enzymes or simply DNAzymes are single-stranded oligo-deoxyribonucleotide molecules that, like proteins and ribozymes, possess the ability to perform catalysis. Although DNAzymes have not yet been found in living organisms, they have been isolated in the laboratory through in vitro selection. The selected DNAzyme sequences have the ability to catalyze a broad range of chemical reactions, utilizing DNA, RNA, peptides or small organic compounds as substrates. DNAmoreDB is a comprehensive database resource for DNAzymes that collects and organizes the following types of information: sequences, conditions of the selection procedure, catalyzed reactions, kinetic parameters, substrates, cofactors, structural information whenever available, and literature references. Currently, DNAmoreDB contains information about DNAzymes that catalyze 20 different reactions. We included a submission form for new data, a REST-based API system that allows users to retrieve the database contents in a machine-readable format, and keyword and BLASTN search features. The database is publicly available at https://www.genesilico.pl/DNAmoreDB/.