Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

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.

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.

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.

Remnants of an Ancient Metabolism without Phosphate.

Phosphate is essential for all living systems, serving as a building block of genetic and metabolic machinery. However, it is unclear how phosphate could have assumed these central roles on primordial Earth, given its poor geochemical accessibility. We used systems biology approaches to explore the alternative hypothesis that a protometabolism could have emerged prior to the incorporation of phosphate. Surprisingly, we identified a cryptic phosphate-independent core metabolism producible from simple prebiotic compounds. This network is predicted to support the biosynthesis of a broad category of key biomolecules. Its enrichment for enzymes utilizing iron-sulfur clusters, and the fact that thermodynamic bottlenecks are more readily overcome by thioester rather than phosphate couplings, suggest that this network may constitute a "metabolic fossil" of an early phosphate-free nonenzymatic biochemistry. Our results corroborate and expand previous proposals that a putative thioester-based metabolism could have predated the incorporation of phosphate and an RNA-based genetic system. PAPERCLIP.

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.

CDK5-cyclin B1 regulates mitotic fidelity.

CDK1 has been known to be the sole cyclin-dependent kinase (CDK) partner of cyclin B1 to drive mitotic progression1. Here we demonstrate that CDK5 is active during mitosis and is necessary for maintaining mitotic fidelity. CDK5 is an atypical CDK owing to its high expression in post-mitotic neurons and activation by non-cyclin proteins p35 and p392. Here, using independent chemical genetic approaches, we specifically abrogated CDK5 activity during mitosis, and observed mitotic defects, nuclear atypia and substantial alterations in the mitotic phosphoproteome. Notably, cyclin B1 is a mitotic co-factor of CDK5. Computational modelling, comparison with experimentally derived structures of CDK-cyclin complexes and validation with mutational analysis indicate that CDK5-cyclin B1 can form a functional complex. Disruption of the CDK5-cyclin B1 complex phenocopies CDK5 abrogation in mitosis. Together, our results demonstrate that cyclin B1 partners with both CDK5 and CDK1, and CDK5-cyclin B1 functions as a canonical CDK-cyclin complex to ensure mitotic fidelity.

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.

Learning from the worm: the effectiveness of protein-bound Moco to treat Moco deficiency.

Molybdenum cofactor (Moco) is synthesized endogenously in humans and is essential for human development. Supplementation of Moco or its precursors has been explored as a therapy to treat Moco-deficient patients but with significant limitations. By using the nematode C. elegans as a model, Warnhoff and colleagues (pp. 212-217) describe the beneficial impact of protein-bound Moco supplementation to treat Moco deficiency. If such an effect is conserved, this advance from basic research in worms may have significant clinical implications as a novel therapy for molybdenum cofactor deficiency.

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a Celegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance.

SUMO modification regulates diverse cellular processes by targeting hundreds of proteins. However, the limited number of sumoylation enzymes raises the question of how such a large number of substrates are efficiently modified. Specifically, how genome maintenance factors are dynamically sumoylated at DNA replication and repair sites to modulate their functions is poorly understood. Here, we demonstrate a role for the conserved yeast Esc2 protein in this process by acting as a SUMO E2 cofactor. Esc2 is required for genome stability and binds to Holliday junctions and replication fork structures. Our targeted screen found that Esc2 promotes the sumoylation of a Holliday junction dissolution complex and specific replisome proteins. Esc2 does not elicit these effects via stable interactions with substrates or their common SUMO E3. Rather, we show that a SUMO-like domain of Esc2 stimulates sumoylation by exploiting a noncovalent SUMO binding site on the E2 enzyme. This role of Esc2 in sumoylation is required for Holliday junction clearance and genome stability. Our findings thus suggest that Esc2 acts as a SUMO E2 cofactor at distinct DNA structures to promote the sumoylation of specific substrates and genome maintenance.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

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.

A high-resolution protein architecture of the budding yeast genome.

The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.

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.

Coevolution of RNA and protein subunits in RNase P and RNase MRP, two RNA processing enzymes.

RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.

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.