The 50 most-cited articles in Biochemistry, 2016

Cholesterol is an essential component of cell membranes and the precursor for the synthesis of steroid hormones and bile acids. The synthesis of this molecule occurs partially in a membranous world (especially the last steps), where the enzymes, substrates, and products involved tend to be extremely hydrophobic. The importance of cholesterol has increased in the past half-century because of its association with cardiovascular diseases, which are considered one of the leading causes of death worldwide. In light of the current need for new drugs capable of controlling the levels of cholesterol in the bloodstream, it is important to understand how cholesterol is synthesized in the organism and identify the main enzymes involved in this process. Taking this into account, this review presents a detailed description of several enzymes involved in the biosynthesis of cholesterol. In this regard, the structure and catalytic mechanism of the enzymes involved in cholesterol biosynthesis, from the initial two-carbon acetyl-CoA building block, will be reviewed and their current pharmacological…

One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp(‡) that are in general negative. That is, the heat capacity (Cp) for the enzyme-substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp(‡) is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp(‡) has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the…

Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral…

Aspirin and other nonsteroidal anti-inflammatory drugs target the cyclooxygenase enzymes (COX-1 and COX-2) to block the formation of prostaglandins. Aspirin is unique in that it covalently modifies each enzyme by acetylating Ser-530 within the cyclooxygenase active site. Acetylation of COX-1 leads to complete loss of activity, while acetylation of COX-2 results in the generation of the monooxygenated product 15(R)-hydroxyeicosatetraenoic acid (15R-HETE). Ser-530 has also been shown to influence the stereochemistry for the addition of oxygen to the prostaglandin product. We determined the crystal structures of S530T murine (mu) COX-2, aspirin-acetylated human (hu) COX-2, and huCOX-2 in complex with salicylate to 1.9, 2.0, and 2.4 Å, respectively. The structures reveal that (1) the acetylated Ser-530 completely blocks access to the hydrophobic groove, (2) the observed binding pose of salicylate is reflective of the enzyme-inhibitor complex prior to acetylation, and (3) the observed Thr-530 rotamer in the S530T muCOX-2 crystal structure does not impede access to the hydrophobic groove.…

Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987-2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed "RosettaScripts" for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta's ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and…

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ(1-40) and Aβ(1-42) into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ(1-42) compared to that of Aβ(1-40) are poorly understood. To explore in detail the structural propensity of the monomeric Aβ(1-40) and Aβ(1-42) peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ(1-40) and Aβ(1-42) peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings ((3)JHNHα, (3)JC'C', (3)JC'Hα, (1)JHαCα, (2)JNCα, and (1)JNCα)…

The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that includes more than 100000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-Acetyltransferases transfer an acetyl group from acetyl-CoA to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur at two different positions, either at the amino-terminal end (αN-acetylation) or at the ε-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups on the basis of their substrate specificity, and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics, and their mechanism of action.

Formate may become an ideal mediator between the physicochemical and biological realms, as it can be produced efficiently from multiple available sources, such as electricity and biomass, and serve as one of the simplest organic compounds for providing both carbon and energy to living cells. However, limiting the realization of formate as a microbial feedstock is the low diversity of formate-fixing enzymes and thereby the small number of naturally occurring formate-assimilation pathways. Here, the natural enzymes and pathways supporting formate assimilation are presented and discussed together with proposed synthetic routes that could permit growth on formate via existing as well as novel formate-fixing reactions. By considering such synthetic routes, the diversity of metabolic solutions for formate assimilation can be expanded dramatically, such that different host organisms, cultivation conditions, and desired products could be matched with the most suitable pathway. Astute application of old and new formate-assimilation pathways may thus become a cornerstone in the development of…

The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods that provide significant insight into the correlation of structure with function have now been developed. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III –OOH non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV ═O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Finally, for several subclasses of non-heme Fe enzymes, binding of the substrate to the Fe II site leads to the one-electron reductive activation of O 2 to an Fe III -superoxide capable of H atom abstraction and…

DNA self-assembling nanostructure has been considered as a promising candidate as a drug delivery vehicle because of its compactness, mechanical stability, and noncytotoxicity. In this work, we developed functional, multiform DNA nanostructures by appending a tumor-penetrating peptide to tetrahedral DNA nanostructure (p-TDN). This functional structure is able to efficiently increase the rate of uptake of glioblastoma cell U87MG compared with the DNA tetrahedron and the double-stranded DNA structures. We found that the DNA tetrahedron plays the main role in the endocytosis of U87MG cells, whereas the tumor-penetrating peptide could also bind to transmembrane glycoprotein neuropilin-1 and mediate the endocytosis of the p-TDN nanostructure. Moreover, given the high efficiency of the growth inhibitory effect of the p-TDN loading doxorubicin hydrochloride, the p-TDN distinguishes itself as a promising candidate as an effective delivery carrier.

Ferredoxins play an important role as an electron donor in iron-sulfur (Fe-S) cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron-sulfur cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron-sulfur cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe-S cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does. The reduced form of each…

The dynamic structure of histones and DNA, also known as chromatin, is regulated by two classes of enzymes: those that mediate covalent modifications on either histone proteins or DNA and those that use the energy generated by ATP hydrolysis to mechanically alter chromatic structure. Both classes of enzymes are often found in large protein complexes. In this review, we describe two such complexes: polycomb repressive complex 2 (PRC2), with the protein methyltransferase EZH2 as its catalytic subunit, and the ATP-dependent chromatin remodeler switch/sucrose non-fermentable (SWI/SNF). EZH2 catalyzes the methylation of lysine 27 on histone H3, a covalent chromatin modification that is associated with repressed heterochromatin. The catalytic activity of SWI/SNF, in contrast, leads to a state of open chromatin associated with active transcription. In this review, we discuss the biochemical properties of both complexes, outline the principles of their regulation, and describe their opposing roles in normal development, which can be perturbed in disease settings such as cancer.

An intriguing consequence of ongoing riboswitch discovery efforts is the occasional identification of metabolic or toxicity response pathways for unusual ligands. Recently, we reported the experimental validation of three distinct bacterial riboswitch classes that regulate gene expression in response to the selective binding of a guanidinium ion. These riboswitch classes, called guanidine-I, -II, and -III, regulate numerous genes whose protein products include previously misannotated guanidine exporters and enzymes that degrade guanidine via an initial carboxylation reaction. Guanidine is now recognized as the primal substrate of many multidrug efflux pumps that are important for bacterial resistance to certain antibiotics. Guanidine carboxylase enzymes had long been annotated as urea carboxylase enzymes but are now understood to participate in guanidine degradation. Herein, we report the existence of a fourth riboswitch class for this ligand, called guanidine-IV. Members of this class use a novel aptamer to selectively bind guanidine and use an unusual expression platform…

Heme is an iron-containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was performed to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK 293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in the level of hemoglobinization relative to that of untreated cells. In vitro heme transfer experiments showed that purified…

[NiFe]-hydrogenases catalyze the reversible conversion of hydrogen gas into protons and electrons and are vital metabolic components of many species of bacteria and archaea. At the core of this enzyme is a sophisticated catalytic center comprising nickel and iron, as well as cyanide and carbon monoxide ligands, which is anchored to the large hydrogenase subunit through cysteine residues. The production of this multicomponent active site is accomplished by a collection of accessory proteins and can be divided into discrete stages. The iron component is fashioned by the proteins HypC, HypD, HypE, and HypF, which functionalize iron with cyanide and carbon monoxide. Insertion of the iron center signals to the metallochaperones HypA, HypB, and SlyD to selectively deliver the nickel to the active site. A specific protease recognizes the completed metal cluster and then cleaves the C-terminus of the large subunit, resulting in a conformational change that locks the active site in place. Finally, the large subunit associates with the small subunit, and the complete holoenzyme translocates…

Amyloid formation by islet amyloid polypeptide (IAPP) contributes to β-cell dysfunction in type 2 diabetes. Perturbation of the β-cell membrane may contribute to IAPP-induced toxicity. We examine the effects of lipid composition, salt, and buffer on IAPP amyloid formation and on the ability of IAPP to induce leakage of model membranes. Even low levels of anionic lipids promote amyloid formation and membrane permeabilization. Increasing the percentage of the anionic lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) or 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol), enhances the rate of amyloid formation and increases the level of membrane permeabilization. The choice of zwitterionic lipid has no noticeable effect on membrane-catalyzed amyloid formation but in most cases affects leakage, which tends to decrease in the following order: 1,2-dioleoyl-sn-glycero-3-phosphocholine > 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine > sphingomyelin. Uncharged lipids that increase the level of membrane order weaken the ability of IAPP to induce leakage. Leakage is due…

The free radical nitric oxide (NO(•)) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-Nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO(•)-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, whereas the reducing agent dithiothreitol (DTT) restored activity, suggesting that catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and trinitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific…

G-Quadruplex and i-motif are tetraplex structures that may form in opposite strands at the same location of a duplex DNA. Recent discoveries have indicated that the two tetraplex structures can have conflicting biological activities, which poses a challenge for cells to coordinate. Here, by performing innovative population analysis on mechanical unfolding profiles of tetraplex structures in double-stranded DNA, we found that formations of G-quadruplex and i-motif in the two complementary strands are mutually exclusive in a variety of DNA templates, which include human telomere and promoter fragments of hINS and hTERT genes. To explain this behavior, we placed G-quadruplex- and i-motif-hosting sequences in an offset fashion in the two complementary telomeric DNA strands. We found simultaneous formation of the G-quadruplex and i-motif in opposite strands, suggesting that mutual exclusivity between the two tetraplexes is controlled by steric hindrance. This conclusion was corroborated in the BCL-2 promoter sequence, in which simultaneous formation of two tetraplexes was observed due to…

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein-protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with…

Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the…

The CopC proteins are periplasmic copper binding proteins believed to play a role in bacterial copper homeostasis. Previous studies have focused on CopCs that are part of seven-protein Cop or Pco systems involved in copper resistance. These canonical CopCs contain distinct Cu(I) and Cu(II) binding sites. Mounting evidence suggests that CopCs are more widely distributed, often present only with the CopD inner membrane protein, frequently as a fusion protein, and that the CopC and CopD proteins together function in the uptake of copper to the cytoplasm. In the methanotroph Methylosinus trichosporium OB3b, genes encoding a CopCD pair are located adjacent to the particulate methane monooxygenase (pMMO) operon. The CopC from this organism (Mst-CopC) was expressed, purified, and structurally characterized. The 1.46 Å resolution crystal structure of Mst-CopC reveals a single Cu(II) binding site with coordination somewhat different from that in canonical CopCs, and the absence of a Cu(I) binding site. Extensive bioinformatic analyses indicate that the majority of CopCs in fact contain only…

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and…

The newly isolated Clostridium beijerinckii [FeFe]-hydrogenase CbA5H was characterized by Fourier transform infrared spectroscopy coupled to enzymatic activity assays. This showed for the first time that in this enzyme the oxygen-sensitive active state H ox can be simply and reversibly converted to the oxygen-stable inactive H inact state. This suggests that oxygen sensitivity is not an intrinsic feature of the catalytic center of [FeFe]-hydrogenases (H-cluster), opening new challenging perspectives on the oxygen sensitivity mechanism as well as new possibilities for exploitation in industrial applications.

The encapsulation of enzymes and other proteins within a proteinaceous shell has been observed in many bacteria and archaea, but the function and utility of many such compartments are enigmatic. Efforts to study these functions have been complicated by the size and complexity of traditional protein compartments. One potential system for investigating the effect of compartmentalization is encapsulin, a large and newly discovered class of protein shells that are typically composed of two proteins: a protomer that assembles into the icosahedral shell and a cargo protein packaged inside. Encapsulins are some of the simplest known protein shell systems and readily self-assemble in vivo. Systematic characterization of the effects of compartmentalization requires the ability to load a wide range of cargo proteins. Here, we demonstrate that foreign cargo can be loaded into the encapsulin from Thermotoga maritima both in vivo and in vitro by fusion of the cargo protein with a short C-terminal peptide present in the native cargo. To facilitate biochemical characterization, we also develop a…

Recently, it was determined that representatives of the riboswitch candidates called ykkC and mini-ykkC directly bind free guanidine. These riboswitches regulate the expression of genes whose protein products are implicated in overcoming the toxic effects of this ligand. Thus, the relevant ykkC motif and mini-ykkC motif RNAs have been classified as guanidine-I and guanidine-II riboswitch RNAs, respectively. Moreover, we had previously noted that a third candidate riboswitch class, called ykkC-III, was associated with a distribution of genes similar to those of the other two motifs. Therefore, it was predicted that ykkC-III motif RNAs would sense and respond to the same ligand. In this report, we present biochemical data supporting the hypothesis that ykkC-III RNAs represent a third class of guanidine-sensing RNAs called guanidine-III riboswitches. Members of the guanidine-III riboswitch class bind their ligand with an affinity similar to that observed for members of the other two classes. Notably, there are some sequence similarities between guanidine-II and guanidine-III…

In 1964, Alfrey and colleagues proposed that acetylation and methylation of histones may regulate RNA synthesis and described "the possibility that relatively minor modifications of histone structure, taking place on the intact protein molecule, offer a means of switching-on or off RNA synthesis at different loci along the chromosome" [Allfrey, V., Faulkner, R., and Mirsky, A. (1964) Proc. Natl. Acad. Sci. U.S.A. 51, 786]. Fifty years later, this prescient description provides a simple but conceptually accurate model for the biological role of histone post-translational modifications (PTMs). The basic unit of chromosomes is the nucleosome, with double-stranded DNA wrapped around a histone protein oligomer. The "tails" of histone proteins are post-translationally modified, which alters the physical properties of nucleosomes in a manner that impacts gene accessibility for transcription and replication. Enzymes that catalyze the addition and removal of histone PTMs, histone-modifying enzymes (HMEs), are present in large protein complexes, with DNA-binding proteins, ATP-dependent…

The MST1 and MST2 protein kinases comprise the GCK-II subfamily of protein kinases. In addition to their amino-terminal kinase catalytic domain, related to that of the Saccharomyces cerevisiae protein kinase Ste20, their most characteristic feature is the presence near the carboxy terminus of a unique helical structure called a SARAH domain; this segment allows MST1/MST2 to homodimerize and to heterodimerize with the other polypeptides that contain SARAH domains, the noncatalytic polypeptides RASSF1-6 and Sav1/WW45. Early studies emphasized the potent ability of MST1/MST2 to induce apoptosis upon being overexpressed, as well as the conversion of the endogenous MST1/MST2 polypeptides to constitutively active, caspase-cleaved catalytic fragments during apoptosis initiated by any stimulus. Later, the cleaved, constitutively active form of MST1 was identified in nonapoptotic, quiescent adult hepatocytes as well as in cells undergoing terminal differentiation, where its presence is necessary to maintain those cellular states. The physiologic regulation of full length MST1/MST2 is…

Fe(II) and α-ketoglutarate-dependent fat mass and obesity associated protein (FTO)-dependent demethylation of m⁶A is important for regulation of mRNA splicing and adipogenesis. Developing FTO-specific inhibitors can help probe the biology of FTO and unravel novel therapeutic targets for treatment of obesity or obesity-associated diseases. In the present paper, we have identified that 4-chloro-6-(6'-chloro-7'-hydroxy-2',4',4'-trimethyl-chroman-2'-yl)benzene-1,3-diol (CHTB) is an inhibitor of FTO. The crystal structure of CHTB complexed with human FTO reveals that the novel small molecule binds to FTO in a specific manner. The identification of the novel small molecule offers opportunities for further development of more selective and potent FTO inhibitors.

Enzymatic installation of chlorine/bromine into unactivated carbon centers provides a versatile, selective, and environmentally friendly alternative to chemical halogenation. Iron(II) and 2-(oxo)-glutarate (Fe II /2OG)-dependent halogenases are powerful biocatalysts that are capable of cleaving aliphatic C–H bonds to introduce useful functional groups, including halogens. Using the structure of the Fe/2OG halogenase, WelO5, in complex with its small molecule substrate, we identified a similar N -acyl amino acid hydroxylase, SadA, and reprogrammed it to halogenate its substrate, thereby generating a new chiral haloalkyl center. The work highlights the potential of Fe II /2OG enzymes as platforms for development of novel stereospecific catalysts for late-stage C–H functionalization.

Substantial attention has recently been devoted to G protein-biased agonism of the μ-opioid receptor (MOR) as an ideal new mechanism for the design of analgesics devoid of serious side effects. However, designing opioids with appropriate efficacy and bias is challenging because it requires an understanding of the ligand binding process and of the allosteric modulation of the receptor. Here, we investigated these phenomena for TRV-130, a G protein-biased MOR small-molecule agonist that has been shown to exert analgesia with less respiratory depression and constipation than morphine and that is currently being evaluated in human clinical trials for acute pain management. Specifically, we carried out multimicrosecond, all-atom molecular dynamics (MD) simulations of the binding of this ligand to the activated MOR crystal structure. Analysis of >50 μs of these MD simulations provides insights into the energetically preferred binding pathway of TRV-130 and its stable pose at the orthosteric binding site of MOR. Information transfer from the TRV-130 binding pocket to the intracellular…

Although the terms "excessive reactive oxygen species (ROS)" and "oxidative stress" are widely used, the implications of oxidative stress are often misunderstood. ROS are not a single species but a variety of compounds, each with unique biochemical properties and abilities to react with biomolecules. ROS cause activation of growth signals through thiol oxidation and may lead to DNA damage at elevated levels. In this review, we first discuss a conceptual framework for the interplay of ROS and antioxidants. This review then describes ROS signaling using FLT3-mediated growth signaling as an example. We then focus on ROS-mediated DNA damage. High concentrations of ROS result in various DNA lesions, including 8-oxo-7,8-dihydro-guanine, oxazolone, DNA-protein cross-links, and hydantoins, that have unique biological impacts. Here we delve into the biochemistry of nine well-characterized DNA lesions. Within each lesion, the types of repair mechanisms, the mutations induced, and their effects on transcription and replication are discussed. Finally, this review will discuss biochemically…

Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher…

Carotenes and their oxygenated derivatives, xanthophylls, are structural elements of the photosynthetic apparatus and contribute to increasing both the light-harvesting and photoprotective capacity of the photosystems. β-Carotene is present in both the core complexes and light-harvesting system (LHCI) of Photosystem (PS) I, while xanthophylls lutein and violaxanthin bind exclusively to its antenna moiety; another xanthophyll, zeaxanthin, which protects chloroplasts against photooxidative damage, binds to the LHCI complexes under conditions of excess light. We functionally dissected various components of the xanthophyll- and carotene-dependent photoprotection mechanism of PSI by analyzing two Arabidopsis mutants: szl1 plants, with a carotene content lower than that of the wild type, and npq1, with suppressed zeaxanthin formation. When exposed to excess light, the szl1 genotype displayed PSI photoinhibition stronger than that of wild-type plants, while removing zeaxanthin had no such effect. The PSI-LHCI complex purified from szl1 was more photosensitive than the corresponding…

The opacity of mammalian tissue to visible light and the strong attenuation of infrared light by water at ≥900 nm have contributed to growing interest in the development of far-red and near-infrared absorbing tools for visualizing and actuating responses within live cells. Here we report the discovery of cyanobacteriochromes (CBCRs) responsive to light in this far-red window. CBCRs are linear tetrapyrrole (bilin)-based light sensors distantly related to plant phytochrome sensors. Our studies reveal far-red (λmax = 725-755 nm)/orange (λmax = 590-600 nm) and far-red/red (λmax = 615-685 nm) photoswitches that are small (<200 amino acids) and can be genetically reconstituted in living cells. Phylogenetic analysis and characterization of additional CBCRs demonstrated that far-red/orange CBCRs evolved after a complex transition from green/red CBCRs known for regulating complementary chromatic acclimation. Incorporation of different bilin chromophores demonstrated that tuning mechanisms responsible for red-shifted chromophore absorption act at the A-, B-, and/or C-rings, whereas…

Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is an essential acyltransferase that mediates flux through plant phenylpropanoid metabolism by catalyzing a reaction between p-coumaroyl-CoA and shikimate, yet it also exhibits broad substrate permissiveness in vitro. How do enzymes like HCT avoid functional derailment by cellular metabolites that qualify as non-native substrates? Here, we combine X-ray crystallography and molecular dynamics to reveal distinct dynamic modes of HCT under native and non-native catalysis. We find that essential electrostatic and hydrogen-bonding interactions between the ligand and active site residues, permitted by active site plasticity, are elicited more effectively by shikimate than by other non-native substrates. This work provides a structural basis for how dynamic conformational states of HCT favor native over non-native catalysis by reducing the number of futile encounters between the enzyme and shikimate.

Fusarium head blight is a plant disease with significant agricultural and health impact which affects cereal crops such as wheat, barley, and maize and is characterized by reduced grain yield and the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). Studies have identified trichothecene production as a virulence factor in Fusarium graminearum and have linked DON resistance to the ability to form DON-3-O-glucoside in wheat. Here, the structures of a deoxynivalenol:UDP-glucosyltransferase (Os79) from Oryza sativa are reported in complex with UDP in an open conformation, in complex with UDP in a closed conformation, and in complex with UDP-2-fluoro-2-deoxy-d-glucose and trichothecene at 1.8, 2.3, and 2.2 Å resolution, respectively. The active site of Os79 lies in a groove between the N-terminal acceptor and the C-terminal donor-binding domains. Structural alignments reveal that Os79 likely utilizes a catalytic mechanism similar to those of other plant UGTs, with His 27 activating the trichothecene O3 hydroxyl for nucleophilic attack at C1' of the UDP-glucose donor.…

In oxygenic photosynthesis, water is oxidized and dioxygen is produced at a Mn4Ca complex bound to the proteins of photosystem II (PSII). Valence and coordination changes in its catalytic S-state cycle are of great interest. In room-temperature (in situ) experiments, time-resolved energy-sampling X-ray emission spectroscopy of the Mn Kβ1,3 line after laser-flash excitation of PSII membrane particles was applied to characterize the redox transitions in the S-state cycle. The Kβ1,3 line energies suggest a high-valence configuration of the Mn4Ca complex with Mn(III)3Mn(IV) in S0, Mn(III)2Mn(IV)2 in S1, Mn(III)Mn(IV)3 in S2, and Mn(IV)4 in S3 and, thus, manganese oxidation in each of the three accessible oxidizing transitions of the water-oxidizing complex. There are no indications of formation of a ligand radical, thus rendering partial water oxidation before reaching the S4 state unlikely. The difference spectra of both manganese Kβ1,3 emission and K-edge X-ray absorption display different shapes for Mn(III) oxidation in the S2 → S3 transition when compared to Mn(III) oxidation in the…

A recently discovered class of endogenous mammalian lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), possesses anti-diabetic and anti-inflammatory activities. Here, we identified and validated carboxyl ester lipase (CEL), a pancreatic enzyme hydrolyzing cholesteryl esters and other dietary lipids, as a FAHFA hydrolase. Variants of CEL have been linked to maturity-onset diabetes of the young, type 8 (MODY8), and to chronic pancreatitis. We tested the FAHFA hydrolysis activity of the CEL MODY8 variant and found a modest increase in activity as compared with that of the normal enzyme. Together, the data suggest that CEL might break down dietary FAHFAs.

The sequence and functional diversity of enzyme superfamilies have expanded through billions of years of evolution from a common ancestor. Understanding how protein sequence and functional "space" have expanded, at both the evolutionary and molecular level, is central to biochemistry, molecular biology, and evolutionary biology. Integrative approaches that examine protein sequence, structure, and function have begun to provide comprehensive views of the functional diversity and evolutionary relationships within enzyme superfamilies. In this review, we outline the recent advances in our understanding of enzyme evolution and superfamily functional diversity. We describe the tools that have been used to comprehensively analyze sequence relationships and to characterize sequence and function relationships. We also highlight recent large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies. We identify several intriguing insights from this recent body of work. First, promiscuous activities are prevalent among extant enzymes.…

The amino acid histidine can play a significant role in the structure and function of proteins. Its various functions include enzyme catalysis, metal binding activity, and involvement in cation-π, π-π, salt-bridge, and other types of noncovalent interactions. Although histidine's imidazole nitrogens (Nδ and Nε) are known to participate in hydrogen bond (HB) interactions as an acceptor or a donor, a systematic study of N-H···N HBs with the Nδ/Nε atom as the acceptor has not been conducted. In this study, we have examined two data sets of ultra-high-resolution (data set I) and very high-resolution (data set II) protein structures and identified 28 and 4017 examples of HBs of the N-H···Nδ/Nε type from both data sets involving histidine imidazole nitrogen as the acceptor. In nearly 70% of them, the main-chain N-H bond is the HB donor, and a majority of the examples are from the N-H group separated by two residues (Ni+2-Hi+2) from histidine. Quantum chemical calculations using model compounds were performed with imidazole and N-methylacetamide, and they assumed conformations from 19…

The pathway from simple abiotically made organic compounds to the molecular bricks of life, as we know it, is unknown. The most efficient geological abiotic route to organic compounds results from the aqueous dissolution of olivine, a reaction known as serpentinization (Sleep, N.H., et al. (2004) Proc. Natl. Acad. Sci. USA 101, 12818-12822). In addition to molecular hydrogen and a reducing environment, serpentinization reactions lead to high-pH alkaline brines that can become easily enriched in silica. Under these chemical conditions, the formation of self-assembled nanocrystalline mineral composites, namely silica/carbonate biomorphs and metal silicate hydrate (MSH) tubular membranes (silica gardens), is unavoidable (Kellermeier, M., et al. In Methods in Enzymology, Research Methods in Biomineralization Science (De Yoreo, J., Ed.) Vol. 532, pp 225-256, Academic Press, Burlington, MA). The osmotically driven membranous structures have remarkable catalytic properties that could be operating in the reducing organic-rich chemical pot in which they form. Among one-carbon compounds,…

The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which…

Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.

Pyruvate formate-lyase (PFL) is a ubiquitous enzyme that supports increased ATP yield during sugar fermentation. While the PFL reaction is known to be reversible in vitro, the ability of PFL to support microbial growth by condensing acetyl-CoA and formate in vivo has never been directly tested. Here, we employ Escherichia coli mutant strains that cannot assimilate acetate via the glyoxylate shunt and use carbon labeling experiments to unequivocally demonstrate PFL-dependent co-assimilation of acetate and formate. Moreover, PFL-dependent growth is faster than growth on acetate using the glyoxylate shunt. Hence, growth via the reverse activity of PFL could have substantial ecological and biotechnological significance.

Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We…

The proto-oncogene PTPN11 encodes a cytoplasmic protein tyrosine phosphatase, SHP2, which is required for normal development and sustained activation of the Ras-MAPK signaling pathway. Germline mutations in SHP2 cause developmental disorders, and somatic mutations have been identified in childhood and adult cancers and drive leukemia in mice. Despite our knowledge of the PTPN11 variations associated with pathology, the structural and functional consequences of many disease-associated mutants remain poorly understood. Here, we combine X-ray crystallography, small-angle X-ray scattering, and biochemistry to elucidate structural and mechanistic features of three cancer-associated SHP2 variants harboring single point mutations within the N-SH2:PTP interdomain autoinhibitory interface. Our findings directly compare the impact of each mutation on autoinhibition of the phosphatase and advance the development of structure-guided and mutation-specific SHP2 therapies.

The oxygen-evolving complex (OEC) of photosystem II has been studied in the S3 state by electron paramagnetic resonance, extended X-ray absorption fine structure (EXAFS), and femtosecond X-ray diffraction (XRD). However, the actual structure of the OEC in the S3 state has yet to be established. Here, we apply hybrid quantum mechanics/molecular mechanics methods and propose a structural model that is consistent with EXAFS and XRD. The model supports binding of water ligands to the cluster in the S2 → S3 transition through a carousel rearrangement around Mn4, inspired by studies of ammonia binding.

Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy ( Fimland, N. et al. ( 2008 ) , Biochim. Biophys. Acta 1784 , 1711 - 1719 ), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix-helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that…

Phenol-soluble modulins (PSMs) are peptide virulence factors produced by staphylococci. These peptides contribute to the overall pathogenicity of these bacteria, eliciting multiple immune responses from host cells. Many of the α-type PSMs exhibit cytolytic properties and are able to lyse particular eukaryotic cells, including erythrocytes, neutrophils, and leukocytes. In addition, they also appear to contribute to the protection of the bacterial cell from the host immune response through biofilm formation and detachment. In this study, three of these peptide toxins, PSMs α1, α3, and β2, normally produced by Staphylococcus aureus, have been synthesized using solid-supported peptide synthesis (SPPS) (PSMα1 and PSMα3) or made by heterologous expression in Escherichia coli (PSMβ2). Their three-dimensional structures were elucidated using nuclear magnetic resonance spectroscopy. PSMα1 and PSMα3 each consist of a single amphipathic helix with a slight bend near the N- and C-termini, respectively. PSMβ2 contains three amphipathic helices, which fold to produce a "v-like" shape between…

The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large…