RESUMO
Bacterial small molecule metabolites such as adenosine-diphosphate-d-glycero-ß-d-manno-heptose (ADP-heptose) and their derivatives act as effective innate immune agonists in mammals. We show that functional nucleotide-diphosphate-heptose biosynthetic enzymes (HBEs) are distributed widely in bacteria, archaea, eukaryotes, and viruses. We identified a conserved STTR5 motif as a hallmark of heptose nucleotidyltransferases that can synthesize not only ADP-heptose but also cytidine-diphosphate (CDP)- and uridine-diphosphate (UDP)-heptose. Both CDP- and UDP-heptoses are agonists that trigger stronger alpha-protein kinase 1 (ALPK1)-dependent immune responses than ADP-heptose in human and mouse cells and mice. We also produced ADP-heptose in archaea and verified its innate immune agonist functions. Hence, the ß-d-manno-heptoses are cross-kingdom, small-molecule, pathogen-associated molecular patterns that activate the ALPK1-dependent innate immune signaling cascade.
Assuntos
Heptoses , Nucleotidiltransferases , Moléculas com Motivos Associados a Patógenos , Animais , Humanos , Camundongos , Motivos de Aminoácidos , Archaea/enzimologia , Bactérias/enzimologia , Bactérias/metabolismo , Heptoses/biossíntese , Heptoses/imunologia , Imunidade Inata , Nucleotidiltransferases/química , Nucleotidiltransferases/classificação , Nucleotidiltransferases/genética , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Proteínas Quinases/metabolismo , Vírus/enzimologiaRESUMO
Alcohol dehydrogenase (ADH) is an important enzyme that catalyzes alcohol oxidation and/or aldehyde reduction. As one of NAD+-dependent ADH types, iron-containing/activated ADH (Fe-ADH) is ubiquitous in Bacteria, Archaea, and Eukaryotes, possessing a similar "tunnel-like" structure that is composed of a domain A in its N-terminus and a domain B in its C-terminus. A conserved "GGGS" sequence in the domain A of Fe-ADH associates with NAD+, and one conserved Asp residue and three conserved His residues in the domain B are its catalytic active sites by surrounding with Fe atom, suggesting that it might employ similar catalytic mechanism. Notably, all the biochemically characterized Fe-ADHs from hyperthermophiles that thrive in above 80 °C possess two unique characteristics that are absent in other Fe-ADHs: thermophilicity and thermostability, thereby demonstrating that they can oxidize alcohol and reduce aldehyde at high temperature. Considering these two unique characteristics, Fe-ADHs from hyperthermophiles are potentially industrial biocatalysts for alcohol and aldehyde biotransformation at high temperature. Herein, we reviewed structural and biochemical characteristics of Fe-ADHs from hyperthermophiles, focusing on similarity and difference between Fe-ADHs from hyperthermophiles and their homologs from non-hyperthermophiles, and between hyperthermophilic archaeal Fe-ADHs and bacterial homologs. Furthermore, we proposed future directions of Fe-ADHs from hyperthermophiles.
Assuntos
Álcool Desidrogenase , Estabilidade Enzimática , Ferro , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Ferro/metabolismo , Ferro/química , Archaea/enzimologia , Domínio Catalítico , Modelos Moleculares , Temperatura Alta , OxirreduçãoRESUMO
Archaea possess characteristic membrane-spanning lipids that are thought to contribute to the adaptation to extreme environments. However, the biosynthesis of these lipids is poorly understood. Here, we identify a radical S-adenosyl-L-methionine (SAM) enzyme that synthesizes glycerol monoalkyl glycerol tetraethers (GMGTs). The enzyme, which we name GMGT synthase (Gms), catalyzes the formation of a C(sp3)-C(sp3) linkage between the two isoprenoid chains of glycerol dialkyl glycerol tetraethers (GDGTs). This conclusion is supported by heterologous expression of gene gms from a GMGT-producing species in a methanogen, as well as demonstration of in vitro activity using purified Gms enzyme. Additionally, we show that genes encoding putative Gms homologs are present in obligate anaerobic archaea and in metagenomes obtained from oxygen-deficient environments, and appear to be absent in metagenomes from oxic settings.
Assuntos
Archaea , Oxigênio , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Archaea/genética , Archaea/metabolismo , Archaea/enzimologia , Oxigênio/metabolismo , Anaerobiose , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Glicerol/metabolismo , Metagenoma , FilogeniaRESUMO
Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.
Assuntos
Archaea , Hidrogênio , Hidrogenase , Filogenia , Archaea/genética , Archaea/enzimologia , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Genoma Arqueal , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Hidrogenase/genética , Hidrogenase/química , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
This review synthesizes recent discoveries of novel archaea clades capable of oxidizing higher alkanes, from volatile ones like ethane to longer-chain alkanes like hexadecane. These archaea, termed anaerobic multicarbon alkane-oxidizing archaea (ANKA), initiate alkane oxidation using alkyl-coenzyme M reductases, enzymes similar to the methyl-coenzyme M reductases of methanogenic and anaerobic methanotrophic archaea (ANME). The polyphyletic alkane-oxidizing archaea group (ALOX), encompassing ANME and ANKA, harbors increasingly complex alkane degradation pathways, correlated with the alkane chain length. We discuss the evolutionary trajectory of these pathways emphasizing metabolic innovations and the acquisition of metabolic modules via lateral gene transfer. Additionally, we explore the mechanisms by which archaea couple alkane oxidation with the reduction of electron acceptors, including electron transfer to partner sulfate-reducing bacteria (SRB). The phylogenetic and functional constraints that shape ALOX-SRB associations are also discussed. We conclude by highlighting the research needs in this emerging research field and its potential applications in biotechnology.
Assuntos
Alcanos , Archaea , Oxirredução , Oxirredutases , Filogenia , Alcanos/metabolismo , Archaea/enzimologia , Archaea/genética , Archaea/metabolismo , Oxirredutases/metabolismo , Oxirredutases/genética , Transporte de Elétrons , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , Transferência Genética Horizontal , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Bactérias/classificaçãoRESUMO
The ParA/MinD (A/D) family of ATPases spatially organize an array of genetic- and protein-based cellular cargos across the bacterial and archaeal domains of life. By far, the two best-studied members, and family namesake, are ParA and MinD, involved in bacterial DNA segregation and divisome positioning, respectively. ParA and MinD make protein waves on the nucleoid or membrane to segregate chromosomes and position the divisome. Less studied is the growing list of A/D ATPases widespread across bacteria and implicated in the subcellular organization of diverse protein-based complexes and organelles involved in myriad biological processes, from metabolism to pathogenesis. Here we describe mechanistic commonality, variation, and coordination among the most widespread family of positioning ATPases used in the subcellular organization of disparate cargos across bacteria and archaea.
Assuntos
Adenosina Trifosfatases , Archaea , Bactérias , Proteínas de Bactérias , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Archaea/genética , Archaea/enzimologia , Archaea/metabolismo , Bactérias/genética , Bactérias/enzimologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Segregação de CromossomosRESUMO
The amide proteogenic amino acids, asparagine and glutamine, are two of the twenty amino acids used in translation by all known life. The aminoacyl-tRNA synthetases for asparagine and glutamine, asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase, evolved after the split in the last universal common ancestor of modern organisms. Before that split, life used two-step indirect pathways to synthesize asparagine and glutamine on their cognate tRNAs to form the aminoacyl-tRNA used in translation. These two-step pathways were retained throughout much of the bacterial and archaeal domains of life and eukaryotic organelles. The indirect routes use non-discriminating aminoacyl-tRNA synthetases (non-discriminating aspartyl-tRNA synthetase and non-discriminating glutamyl-tRNA synthetase) to misaminoacylate the tRNA. The misaminoacylated tRNA formed is then transamidated into the amide aminoacyl-tRNA used in protein synthesis by tRNA-dependent amidotransferases (GatCAB and GatDE). The enzymes and tRNAs involved assemble into complexes known as transamidosomes to help maintain translational fidelity. These pathways have evolved to meet the varied cellular needs across a diverse set of organisms, leading to significant variation. In certain bacteria, the indirect pathways may provide a means to adapt to cellular stress by reducing the fidelity of protein synthesis. The retention of these indirect pathways versus acquisition of asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase in lineages likely involves a complex interplay of the competing uses of glutamine and asparagine beyond translation, energetic costs, co-evolution between enzymes and tRNA, and involvement in stress response that await further investigation.
Assuntos
Aminoacil-tRNA Sintetases , Evolução Molecular , Biossíntese de Proteínas , Aminoacil-RNA de Transferência , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/genética , Asparagina/metabolismo , Asparagina/genética , Glutamina/metabolismo , Bactérias/genética , Bactérias/enzimologia , Bactérias/metabolismo , Archaea/genética , Archaea/metabolismo , Archaea/enzimologia , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Amidas/metabolismo , HumanosRESUMO
The endoribonuclease RNase P is responsible for tRNA 5' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.
Assuntos
Evolução Molecular , Ribonuclease P , Animais , Humanos , Archaea/enzimologia , Archaea/genética , Bactérias/enzimologia , Bactérias/genética , Filogenia , Ribonuclease P/química , Ribonuclease P/classificação , Ribonuclease P/genética , Ribonuclease P/metabolismo , RNA CatalíticoRESUMO
How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.
Assuntos
Aminoacil-tRNA Sintetases , Evolução Molecular , Código Genético , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Bactérias/enzimologia , Bactérias/genética , Filogenia , Archaea/enzimologia , Archaea/genética , Eucariotos/enzimologia , Eucariotos/genéticaRESUMO
Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).
Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.
Assuntos
Aminoacil-tRNA Sintetases , Archaea , Microbioma Gastrointestinal , Humanos , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/isolamento & purificação , Aminoacil-tRNA Sintetases/metabolismo , Archaea/enzimologia , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacilação de RNA de TransferênciaRESUMO
The Enzyme Function Initiative (EFI) provides a web resource with "genomic enzymology" web tools to leverage the protein (UniProt) and genome (European Nucleotide Archive; ENA; https://www.ebi.ac.uk/ena/) databases to assist the assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized enzymes (https://efi.igb.illinois.edu/). The tools enable (1) exploration of sequence-function space in enzyme families using sequence similarity networks (SSNs; EFI-EST), (2) easy access to genome context for bacterial, archaeal, and fungal proteins in the SSN clusters so that isofunctional families can be identified and their functions inferred from genome context (EFI-GNT); and (3) determination of the abundance of SSN clusters in NIH Human Metagenome Project metagenomes using chemically guided functional profiling (EFI-CGFP). We describe enhancements that enable SSNs to be generated from taxonomy categories, allowing higher resolution analyses of sequence-function space; we provide examples of the generation of taxonomy category-specific SSNs.
Assuntos
Bases de Dados Genéticas , Enzimas , Internet , Humanos , Bactérias/enzimologia , Bactérias/genética , Genômica , Metagenoma , Enzimas/química , Enzimas/genética , Archaea/enzimologia , Archaea/genética , Fungos/enzimologia , Fungos/genéticaRESUMO
Family A DNA polymerases (PolAs) form an important and well-studied class of extant polymerases participating in DNA replication and repair. Nonetheless, despite the characterization of multiple subfamilies in independent, dedicated works, their comprehensive classification thus far is missing. We therefore re-examine all presently available PolA sequences, converting their pairwise similarities into positions in Euclidean space, separating them into 19 major clusters. While 11 of them correspond to known subfamilies, eight had not been characterized before. For every group, we compile their general characteristics, examine their phylogenetic relationships and perform conservation analysis in the essential sequence motifs. While most subfamilies are linked to a particular domain of life (including phages), one subfamily appears in Bacteria, Archaea and Eukaryota. We also show that two new bacterial subfamilies contain functional enzymes. We use AlphaFold2 to generate high-confidence prediction models for all clusters lacking an experimentally determined structure. We identify new, conserved features involving structural alterations, ordered insertions and an apparent structural incorporation of a uracil-DNA glycosylase (UDG) domain. Finally, genetic and structural analyses of a subset of T7-like phages indicate a splitting of the 3'-5' exo and pol domains into two separate genes, observed in PolAs for the first time.
Assuntos
Bactérias , DNA Polimerase Dirigida por DNA , Archaea/enzimologia , Bactérias/enzimologia , DNA Polimerase Dirigida por DNA/química , Eucariotos/enzimologia , Filogenia , Uracila-DNA Glicosidase/químicaRESUMO
Ammonia oxidation, as the first step of nitrification, constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase, is lacking, in particular for the environmentally abundant ammonia-oxidizing archaea (AOA). Here the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Besides the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modeling and in-gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase but also reveals clear differences in extracellular domains of the enzyme. These data open avenues for further structure-function studies of this ecologically important nitrification complex.
Assuntos
Archaea , Oxirredutases , Archaea/classificação , Archaea/enzimologia , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Nitrificação , Eletroforese em Gel de Poliacrilamida Nativa , Filogenia , Expressão GênicaRESUMO
Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.
Assuntos
Archaea , Cisteína , Proteínas Ferro-Enxofre , RNA de Transferência , Tiossulfato Sulfurtransferase , Archaea/enzimologia , Archaea/genética , Catálise , Cisteína/metabolismo , Proteínas Ferro-Enxofre/metabolismo , RNA de Transferência/metabolismo , Tiossulfato Sulfurtransferase/química , Tiossulfato Sulfurtransferase/genética , Tiossulfato Sulfurtransferase/metabolismo , Motivos de Aminoácidos , Mutagênese , Domínios Proteicos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismoRESUMO
The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNAPyl) sequences from diverse archaea and bacteria. This analysis suggests that the PylRSâ¢tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.
Assuntos
Aminoacil-tRNA Sintetases , Archaea , Código Genético , Lisina , Aminoacil-tRNA Sintetases/metabolismo , Archaea/enzimologia , Archaea/genética , Lisina/análogos & derivados , Lisina/genética , Methanosarcina , RNA de Transferência/genéticaRESUMO
Enzymes catalyze key reactions within Earth's life-sustaining biogeochemical cycles. Here, we use metaproteomics to examine the enzymatic capabilities of the microbial community (0.2 to 3 µm) along a 5,000-km-long, 1-km-deep transect in the central Pacific Ocean. Eighty-five percent of total protein abundance was of bacterial origin, with Archaea contributing 1.6%. Over 2,000 functional KEGG Ontology (KO) groups were identified, yet only 25 KO groups contributed over half of the protein abundance, simultaneously indicating abundant key functions and a long tail of diverse functions. Vertical attenuation of individual proteins displayed stratification of nutrient transport, carbon utilization, and environmental stress. The microbial community also varied along horizontal scales, shaped by environmental features specific to the oligotrophic North Pacific Subtropical Gyre, the oxygen-depleted Eastern Tropical North Pacific, and nutrient-rich equatorial upwelling. Some of the most abundant proteins were associated with nitrification and C1 metabolisms, with observed interactions between these pathways. The oxidoreductases nitrite oxidoreductase (NxrAB), nitrite reductase (NirK), ammonia monooxygenase (AmoABC), manganese oxidase (MnxG), formate dehydrogenase (FdoGH and FDH), and carbon monoxide dehydrogenase (CoxLM) displayed distributions indicative of biogeochemical status such as oxidative or nutritional stress, with the potential to be more sensitive than chemical sensors. Enzymes that mediate transformations of atmospheric gases like CO, CO2, NO, methanethiol, and methylamines were most abundant in the upwelling region. We identified hot spots of biochemical transformation in the central Pacific Ocean, highlighted previously understudied metabolic pathways in the environment, and provided rich empirical data for biogeochemical models critical for forecasting ecosystem response to climate change.
Assuntos
Proteínas Arqueais , Proteínas de Bactérias , Microbiota , Nitrificação , Água do Mar , Archaea/classificação , Archaea/enzimologia , Proteínas Arqueais/análise , Bactérias/classificação , Bactérias/enzimologia , Proteínas de Bactérias/análise , Biodiversidade , Nitrito Redutases/metabolismo , Oceano Pacífico , Proteômica/métodos , Água do Mar/microbiologiaRESUMO
RNA polymerase III (Pol III) is a large multisubunit complex conserved in all eukaryotes that plays an essential role in producing a variety of short non-coding RNAs, such as tRNA, 5S rRNA and U6 snRNA transcripts. Pol III comprises of 17 subunits in both yeast and human with a 10-subunit core and seven peripheral subunits. Because of its size and complexity, Pol III has posed a formidable challenge to structural biologists. The first atomic cryogenic electron microscopy structure of yeast Pol III leading to the canonical view was reported in 2015. Within the last few years, the optimization of endogenous extract and purification procedure and the technical and methodological advances in cryogenic electron microscopy, together allow us to have a first look at the unprecedented details of human Pol III organization. Here, we look back on the structural studies of human Pol III and discuss them in the light of our current understanding of its role in eukaryotic transcription.
Assuntos
Modelos Moleculares , Conformação Proteica , RNA Polimerase III/química , RNA Polimerase III/metabolismo , Archaea/enzimologia , Sequência Conservada , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , RNA Polimerase III/genética , Relação Estrutura-Atividade , Leveduras/enzimologiaRESUMO
The SKY hot spring is a unique site filled with a thick layer of plant litter. With the advancement of next-generation sequencing, it is now possible to mine many new biocatalyst sequences. In this study, we aimed to (i) identify the metataxonomic of prokaryotes and eukaryotes in microbial mats using 16S and 18S rRNA markers, (ii) and explore carbohydrate degrading enzymes (CAZymes) that have a high potential for future applications. Green microbial mat, predominantly photosynthetic bacteria, was attached to submerged or floating leaves litter. At the spring head, the sediment mixture consisted of plant debris, predominantly brownish-reddish gelatinous microbial mat, pale tan biofilm, and grey-white filament biofilm. The population in the spring head had a higher percentage of archaea and hyperthermophiles than the green mat. Concurrently, we cataloged nearly 10,000 sequences of CAZymes in both green and brown biofilms using the shotgun metagenomic sequencing approach. These sequences include ß-glucosidase, cellulase, xylanase, α-N-arabinofuranosidase, α-L-arabinofuranosidase, and other CAZymes. In conclusion, this work elucidated that SKY is a unique hot spring due to its rich lignocellulosic material, often absent in other hot springs. The data collected from this study serves as a repository of new thermostable macromolecules, in particular families of glycoside hydrolases.
Assuntos
Biofilmes , Carboidratos/química , Lignina/química , Consórcios Microbianos/genética , Archaea/enzimologia , Archaea/genética , Bactérias/química , Bactérias/genética , Biodiversidade , Glicosídeo Hidrolases/química , Temperatura Alta , Metagenoma/genética , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Here, we investigated the impact of different turning frequency (TF) on dynamic changes of N fractions, NH3 emission and bacterial/archaeal community during chicken manure composting. Compared to higher TF (i.e., turning every 1 or 3 days in CMS1 or CMS3 treatments, respectively), lower TF (i.e., turning every 5 or 7 days in CMS5 or CMS7 treatments, respectively) decreased NH3 emission by 11.42-18.95%. Compared with CMS1, CMS3 and CMS7 treatments, the total nitrogen loss of CMS5 decreased by 38.03%, 17.06% and 24.76%, respectively. Ammonia oxidizing bacterial/archaeal (AOB/AOA) communities analysis revealed that the relative abundance of Nitrosospira and Nitrososphaera was higher in lower TF treatment during the thermophilic and cooling stages, which could contribute to the reduction of NH3 emission. Thus, different TF had a great influence on NH3 emission and microbial community during composting. It is practically feasible to increase the abundance of AOB/AOA through adjusting TF and reduce NH3 emission the loss of nitrogen during chicken manure composting.
Assuntos
Amônia/análise , Compostagem/métodos , Esterco/análise , Amônia/química , Animais , Archaea/classificação , Archaea/enzimologia , Bactérias/classificação , Bactérias/enzimologia , Galinhas , Correlação de Dados , Meio Ambiente , Germinação , Concentração de Íons de Hidrogênio , Esterco/microbiologia , Microbiota , Nitratos/análise , Nitrogênio/análise , Nitrogênio/metabolismo , Oxirredução , Caules de Planta , Glycine max , Temperatura , ÁguaRESUMO
Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.