Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Conformational dynamics of adenylate kinase in crystals.

Adenylate kinase is a ubiquitous enzyme in living systems and undergoes dramatic conformational changes during its catalytic cycle. For these reasons, it is widely studied by genetic, biochemical, and biophysical methods, both experimental and theoretical. We have determined the basic crystal structures of three differently liganded states of adenylate kinase from Methanotorrus igneus, a hyperthermophilic organism whose adenylate kinase is a homotrimeric oligomer. The multiple copies of each protomer in the asymmetric unit of the crystal provide a unique opportunity to study the variation in the structure and were further analyzed using advanced crystallographic refinement methods and analysis tools to reveal conformational heterogeneity and, thus, implied dynamic behaviors in the catalytic cycle.

Utilizing a cell-free protein synthesis platform for the biosynthesis of a natural product, caffeine.

Natural products are a valuable source of pharmaceuticals, providing a majority of the small-molecule drugs in use today. However, their production through organic synthesis or in heterologous hosts can be difficult and time-consuming. Therefore, to allow for easier screening and production of natural products, we demonstrated the use of a cell-free protein synthesis system to partially assemble natural products in vitro using S-Adenosyl Methionine (SAM)-dependent methyltransferase enzyme reactions. The tea caffeine synthase, TCS1, was utilized to synthesize caffeine within a cell-free protein synthesis system. Cell-free systems also provide the benefit of allowing the use of substrates that would normally be toxic in a cellular environment to synthesize novel products. However, TCS1 is unable to utilize a compound like S-adenosyl ethionine as a cofactor to create ethylated caffeine analogs. The automation and reduced metabolic engineering requirements of cell-free protein synthesis systems, in combination with other synthesis methods, may enable the more efficient generation of new compounds. Graphical Abstract.

Heterogeneity in M. tuberculosis ß-lactamase inhibition by Sulbactam.

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

A deep learning solution for crystallographic structure determination.

The general de novo solution of the crystallographic phase problem is difficult and only possible under certain conditions. This paper develops an initial pathway to a deep learning neural network approach for the phase problem in protein crystallography, based on a synthetic dataset of small fragments derived from a large well curated subset of solved structures in the Protein Data Bank (PDB). In particular, electron-density estimates of simple artificial systems are produced directly from corresponding Patterson maps using a convolutional neural network architecture as a proof of concept.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

An NmrA-like enzyme-catalysed redox-mediated Diels-Alder cycloaddition with anti-selectivity.

The Diels-Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels-Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels-Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions. Here we report the discovery and characterization of CtdP, a member of a new class of bifunctional oxidoreductase/Diels-Alderase, which was previously annotated as an NmrA-like transcriptional regulator. We demonstrate that CtdP catalyses the inherently disfavoured cycloaddition to form the bicyclo[2.2.2]diazaoctane scaffold with a strict α-anti-selectivity. Guided by computational studies, we reveal a NADP+/NADPH-dependent redox mechanism for the CtdP-catalysed inverse electron demand Diels-Alder cycloaddition, which serves as the first example of a bifunctional Diels-Alderase that utilizes this mechanism.

Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid.

Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of O-methyltyrosine (OMeY). Specifically, we endowed organisms with a marformycins biosynthetic pathway-derived methyltransferase that efficiently converts tyrosine to OMeY in the presence of the co-factor S-adenosylmethionine. The resulting cells can produce and site-specifically incorporate OMeY into proteins at much higher levels than cells exogenously fed OMeY. To understand the structural basis for the substrate selectivity of the transferase, we solved the X-ray crystal structures of the ligand-free and tyrosine-bound enzymes. Most importantly, we have extended this OMeY biosynthetic system to both mammalian cells and the zebrafish model to enhance the utility of genetic code expansion. The creation of autonomous eukaryotes using a 21st amino acid will make genetic code expansion technology more applicable to multicellular organisms, providing valuable vertebrate models for biological and biomedical research.

The Structure of the Arabidopsis PEX4-PEX22 Peroxin Complex-Insights Into Ubiquitination at the Peroxisomal Membrane.

Peroxisomes are eukaryotic organelles that sequester critical oxidative reactions and process the resulting reactive oxygen species into less toxic byproducts. Peroxisome function and formation are coordinated by peroxins (PEX proteins) that guide peroxisome biogenesis and division and shuttle proteins into the lumen and membrane of the organelle. Despite the importance of peroxins in plant metabolism and development, no plant peroxin structures have been reported. Here we report the X-ray crystal structure of the PEX4-PEX22 peroxin complex from the reference plant Arabidopsis thaliana. PEX4 is a ubiquitin-conjugating enzyme (UBC) that ubiquitinates proteins associated with the peroxisomal membrane, and PEX22 is a peroxisomal membrane protein that anchors PEX4 to the peroxisome and facilitates PEX4 activity. We co-expressed Arabidopsis PEX4 as a translational fusion with the soluble PEX4-interacting domain of PEX22 in E. coli. The fusion was linked via a protease recognition site, allowing us to separate PEX4 and PEX22 following purification and solve the structure of the complex. We compared the structure of the PEX4-PEX22 complex to the previously published structures of yeast orthologs. Arabidopsis PEX4 displays the typical UBC structure expected from its sequence. Although Arabidopsis PEX22 lacks notable sequence identity to yeast PEX22, it maintains a similar Rossmann fold-like structure. Several salt bridges are positioned to contribute to the specificity of PEX22 for PEX4 versus other Arabidopsis UBCs, and the long unstructured PEX22 tether would allow PEX4-mediated ubiquitination of distant peroxisomal membrane targets without dissociation from PEX22. The Arabidopsis PEX4-PEX22 structure also revealed that the residue altered in pex4-1 (P123L), a mutant previously isolated via a forward-genetic screen for peroxisomal dysfunction, is near the active site cysteine of PEX4. We demonstrated in vitro UBC activity for the PEX4-PEX22 complex and found that the pex4-1 enzyme has reduced in vitro ubiquitin-conjugating activity and altered specificity compared to PEX4. Our findings illuminate the role of PEX4 and PEX22 in peroxisome structure and function and provide tools for future exploration of ubiquitination at the peroxisome surface.

The crystal structure of DynF from the dynemicin-biosynthesis pathway of Micromonospora chersina.

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded ß-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.

Structure and Function of a Dual Reductase-Dehydratase Enzyme System Involved in p-Terphenyl Biosynthesis.

We report the identification of the ter gene cluster responsible for the formation of the p-terphenyl derivatives terfestatins B and C and echoside B from the Appalachian Streptomyces strain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate. X-ray crystallography of the apo and substrate-bound forms for both enzymes provides additional mechanistic insights. Validation of the TerC structural model via mutagenesis highlights a critical role of arginine 143 and aspartate 173 in catalysis. Cumulatively, this work highlights a set of enzymes acting in harmony to control and direct reactive intermediates and advances fundamental understanding of the previously unresolved early steps in terphenyl biosynthesis.

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography.

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis ß-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Structural characterization of DynU16, a START/Bet v1-like protein involved in dynemicin biosynthesis.

The 1.5 Šresolution crystal structure of DynU16, a protein identified in the dynemicin-biosynthetic gene cluster, is reported. The structure adopts a di-domain helix-grip fold with a uniquely positioned open cavity connecting the domains. The elongated dimensions of the cavity appear to be compatible with the geometry of a linear polyene, suggesting the involvement of DynU16 in the upstream steps of dynemicin biosynthesis.

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins.

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible ß-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

Moving beyond static snapshots: Protein dynamics and the Protein Data Bank.

Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although the dynamics are more difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics, and function. This progress is highlighted in case studies from myoglobin and adenylate kinase to the ribosome and molecular motors where these molecules are being probed with a multitude of techniques across many timescales. New approaches to time-resolved crystallography are allowing simple "movies" to be taken of proteins in action, and new methods of mapping the variations in cryo-electron microscopy are emerging to reveal a more complete description of life's machines. The results of these new methods are aided in their dissemination by continual improvements in curation and distribution by the Protein Data Bank and their partners around the world.

A collagen glucosyltransferase drives lung adenocarcinoma progression in mice.

Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.

Pump-Probe Circular Dichroism Spectroscopy of Cyanobacteriochrome TePixJ Yields: Insights into Its Photoconversion.

The bilin-containing photoreceptor TePixJ, a member of the cyanobacteriochrome (CBCR) family of phytochromes, switches between blue-light-absorbing and green-light-absorbing states in order to drive phototaxis in Thermosynechococcus elongatus. Its photoswitching process involves the formation of a thioether linkage between the C10 carbon of phycoviolobilin and the sidechain of Cys494 during the change in state from green-absorbing to blue-absorbing forms. Complex changes in the binding pocket propagate the signal to other domains for downstream signaling. Here, we report time-resolved circular dichroism experiments in addition to pump-probe absorption measurements for interpretation of the biophysical mechanism of the green-to-blue photoconversion process of this receptor.