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► Histone deacetylases (HDACs) catalyze the hydrolysis of acetyl-
l-lysine. ► HDACs and related deacetylases adopt the α/β arginase fold. ► HDACs employ a promoted-water mechanism for ...catalysis. ► Potent HDAC inhibitors chelate the active site metal ion. ► Macrocyclic HDAC inhibitors interact with the mouth of the active site.
Metal-dependent histone deacetylases (HDACs) catalyze the hydrolysis of acetyl-
l-lysine side chains in histone and nonhistone proteins to yield
l-lysine and acetate. This chemistry plays a critical role in the regulation of numerous biological processes. Aberrant HDAC activity is implicated in various diseases, and HDACs are validated targets for drug design. Two HDAC inhibitors are currently approved for cancer chemotherapy, and other inhibitors are in clinical trials. To date, X-ray crystal structures are available for four human HDACs (2, 4, 7, and 8) and three HDAC-related deacetylases from bacteria (histone deacetylase-like protein (HDLP); histone deacetylase-like amidohydrolase (HDAH); acetylpolyamine amidohydrolase (APAH)). Structural comparisons among these enzymes reveal a conserved constellation of active site residues, suggesting a common mechanism for the metal-dependent hydrolysis of acetylated substrates. Structural analyses of HDACs and HDAC-related deacetylases guide the design of tight-binding inhibitors, and future prospects for developing isozyme-specific inhibitors are quite promising.
Largazole is a macrocyclic depsipeptide originally isolated from the marine cyanobacterium Symploca sp., which is indigenous to the warm, blue-green waters of Key Largo, Florida (whence largazole ...derives its name). Largazole contains an unusual thiazoline–thiazole ring system that rigidifies its macrocyclic skeleton, and it also contains a lipophilic thioester side chain. Hydrolysis of the thioester in vivo yields largazole thiol, which exhibits remarkable antiproliferative effects and is believed to be the most potent inhibitor of the metal-dependent histone deacetylases (HDACs). Here, the 2.14 Å-resolution crystal structure of the HDAC8–largazole thiol complex is the first of an HDAC complexed with a macrocyclic inhibitor and reveals that ideal thiolate–zinc coordination geometry is the key chemical feature responsible for its exceptional affinity and biological activity. Notably, the core structure of largazole is conserved in romidepsin, a depsipeptide natural product formulated as the drug Istodax recently approved for cancer chemotherapy. Accordingly, the structure of the HDAC8–largazole thiol complex is the first to illustrate the mode of action of a new class of therapeutically important HDAC inhibitors.
Ox-/thiazoline groups in nonribosomal peptides are formed by a variant of peptide-forming condensation domains called heterocyclization (Cy) domains and appear in a range of pharmaceutically ...important natural products and virulence factors. Recent cryo-EM, crystallographic, and NMR studies of Cy domains make it opportune to revisit outstanding questions regarding their molecular mechanisms. This review covers structural and dynamical findings about Cy domains that will inform future bioengineering efforts and our understanding of natural product synthesis.
•Heterocyclization domains catalyze both peptide bond formation and cyclodehydration in nonribosomal peptide synthetases.•Heterocyclization domains appear to change conformations for distinct catalytic steps.•Structural dynamics likely couple molecular communication with fold remodeling.•Conformational changes and structural dynamics should be considered in future bioengineering of heterocyclization domains.
Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA–F. A cyclization ...domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and L-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes L-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.
Sulfur is one of the essential elements of life, being involved in pathways to generate sulfide, which is used downstream for amino acid, metabolite, and signaling molecule synthesis. Inorganic ...sulfate and sulfite are the typical sources of sulfur for these bacteria; however, limiting sulfate/sulfite conditions require bacteria to use alternative sulfur sources. Within Pseudomonas fluorescens a set of genes are expressed that allow them to utilize alkanesulfonates to generate sulfite in this limiting condition; the proteins expressed are two‐component flavin‐dependent monooxygenases (TC‐FMOs) involved in the desulfonation of the organosulfur compound and a flavin mononucleotide (FMN) reductase essential for the generation of reduced FMN. The first step in this pathway is SfnG, a TC‐FMO that acts upon dimethylsulfone to produce methanesulfinate, which is further acted on by enzymes in this pathway to generate sulfite. Current work has structurally characterized SfnG through X‐ray crystallography with the ligands FMN and DMSO2 to 1.75‐Å resolution and without ligands to 2.6‐Å resolution. The characterization of SfnG elucidated residues predicted to be important for binding of DMSO2 and FMN within the active site. Further structural analysis showed the active site of SfnG is enclosed by a lid region which is disordered without substrates bound, and the structural arrangement of ligands and the predicted oxygen binding site is in support of SfnG utilizing the recently proposed N5‐peroxyflavin intermediate. Further analysis of SfnG has revealed an interesting quaternary state reliant on substrate binding. The quaternary structure organization is different from other TC‐FMO’s involved in C–S bond cleavage, which was further explored through FMN binding studies. These results will contribute towards our knowledge of TC‐FMOs involved in C–S bond cleavage.
Orthology characterizes genes of different organisms that arose from a single ancestral gene via speciation, in contrast to paralogy, which is assigned to genes that arose via gene duplication. An ...accurate orthology assignment is a crucial step for comparative genomic studies. Orthologous genes in two organisms can be identified by applying a so-called reciprocal search strategy, given that complete information of the organisms' gene repertoire is available. In many investigations, however, only a fraction of the gene content of the organisms under study is examined (e.g., RNA sequencing). Here, identification of orthologous nucleotide or amino acid sequences can be achieved using a graph-based approach that maps nucleotide sequences to genes of known orthology. Existing implementations of this approach, however, suffer from algorithmic issues that may cause problems in downstream analyses.
We present a new software pipeline, Orthograph, that addresses and solves the above problems and implements useful features for a wide range of comparative genomic and transcriptomic analyses. Orthograph applies a best reciprocal hit search strategy using profile hidden Markov models and maps nucleotide sequences to the globally best matching cluster of orthologous genes, thus enabling researchers to conveniently and reliably delineate orthologs and paralogs from transcriptomic and genomic sequence data. We demonstrate the performance of our approach on de novo-sequenced and assembled transcript libraries of 24 species of apoid wasps (Hymenoptera: Aculeata) as well as on published genomic datasets.
With Orthograph, we implemented a best reciprocal hit approach to reference-based orthology prediction for coding nucleotide sequences such as RNAseq data. Orthograph is flexible, easy to use, open source and freely available at https://mptrsen.github.io/Orthograph . Additionally, we release 24 de novo-sequenced and assembled transcript libraries of apoid wasp species.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but ...whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.
Graphical Abstract
Graphical Abstract
Pathways of thymidine hypermodification.
The inherent structural properties of enzymes are critical in defining catalytic function. Often, studies to evaluate the relationship between structure and function are limited to only one defined ...structural element. The two-component flavin-dependent desulfonase family of enzymes involved in bacterial sulfur acquisition utilize a comprehensive range of structural features to carry out the desulfonation of organosulfur compounds. These metabolically essential two-component FMN-dependent desulfonase systems have been proposed to utilize oligomeric changes, protein-protein interactions for flavin transfer, and common mechanistic steps for carbon-sulfur bond cleavage. This review is focused on our current functional and structural understanding of two-component FMN-dependent desulfonase systems from multiple bacterial sources. Mechanistic and structural comparisons from recent independent studies provide fresh insights into the overall functional properties of these systems and note areas in need of further investigation. The review acknowledges current studies focused on evaluating the structural properties of these enzymes in relationship to their distinct catalytic function. The role of these enzymes in maintaining adequate sulfur levels, coupled with the conserved nature of these enzymes in diverse bacteria, underscore the importance in understanding the functional and structural nuances of these systems.
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•Gene organization of enzymes involved in sulfur acquisition.•Structure and function of the two-component FMN-dependent desulfonase systems.•Proposed mechanism of desulfonation by the desulfonase enzymes.•Protein-protein interactions involved in flavin transfer.
Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found ...in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.
AdoMet radical enzymes are involved in processes such as cofactor biosynthesis, anaerobic metabolism, and natural product biosynthesis. These enzymes utilize the reductive cleavage of ...S-adenosylmethionine (AdoMet) to afford l-methionine and a transient 5′-deoxyadenosyl radical, which subsequently generates a substrate radical species. By harnessing radical reactivity, the AdoMet radical enzyme superfamily is responsible for an incredible diversity of chemical transformations. Structural analysis reveals that family members adopt a full or partial Triose-phosphate Isomerase Mutase (TIM) barrel protein fold, containing core motifs responsible for binding a catalytic 4Fe–4S cluster and AdoMet. Here we evaluate over twenty structures of AdoMet radical enzymes and classify them into two categories: ‘traditional’ and ‘ThiC-like’ (named for the structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (ThiC)). In light of new structural data, we reexamine the ‘traditional’ structural motifs responsible for binding the 4Fe–4S cluster and AdoMet, and compare and contrast these motifs with the ThiC case. We also review how structural data combine with biochemical, spectroscopic, and computational data to help us understand key features of this enzyme superfamily, such as the energetics, the triggering, and the molecular mechanisms of AdoMet reductive cleavage. This article is part of a Special Issue entitled: Radical SAM Enzymes and Radical Enzymology.
► Structural motifs for AdoMet radical enzymes are re-examined. ► Structures are classified into two categories: traditional and ThiC-like. ► Recent insights into function through structure are reviewed.
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