Nucleotide Cyclases Research Articles

Bioinformatic analysis of type III CRISPR systems reveals key properties and new effector families.

Recognition of RNA from invading mobile genetic elements (MGE) prompts type III CRISPR systems to activate an HD nuclease domain and/or a nucleotide cyclase domain in the Cas10 subunit, eliciting an immune response. The cyclase domain can generate a range of nucleotide second messengers, which in turn activate a diverse family of ancillary effector proteins. These provide immunity by non-specific degradation of host and MGE nucleic acids or proteins, perturbation of membrane potentials, transcriptional responses, or the arrest of translation. The wide range of nucleotide activators and downstream effectors generates a complex picture that is gradually being resolved. Here, we carry out a global bioinformatic analysis of type III CRISPR loci in prokaryotic genomes, defining the relationships of Cas10 proteins and their ancillary effectors. Our study reveals that cyclic tetra-adenylate is by far the most common signalling molecule used and that many loci have multiple effectors. These typically share the same activator and may work synergistically to combat MGE. We propose four new candidate effector protein families and confirm experimentally that the Csm6-2 protein, a highly diverged, fused Csm6 effector, is a ribonuclease activated by cyclic hexa-adenylate.

Reversible conjugation of a CBASS nucleotide cyclase regulates bacterial immune response to phage infection

Prokaryotic antiviral defence systems are frequently toxic for host cells and stringent regulation is required to ensure survival and fitness. These systems must be readily available in case of infection but tightly controlled to prevent activation of an unnecessary cellular response. Here we investigate how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a type II CBASS system from Bacillus cereus into the model organism Bacillus subtilis, we show that the protein-conjugating Cap2 (CBASS associated protein 2) enzyme links the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. The cyclase–PspA conjugation is reversed by the deconjugating isopeptidase Cap3 (CBASS associated protein 3). We propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage, with conjugation released upon infection, priming the cyclase for activation.

Peptide-Mediated Cyclic Nucleotide Signaling in Plants: Identification and Characterization of Interactor Proteins with Nucleotide Cyclase Activity.

During the last decades, an increasing number of plant signaling peptides have been discovered and it appears that many of them are specific ligands for interacting receptor molecules. These receptors can enable the formation of second messengers which in turn transmit the ligand-induced stimuli into complex and tunable downstream responses. In order to perform such complex tasks, receptor proteins often contain several distinct domains such as a kinase and/or adenylate cyclase (AC) or guanylate cyclase (GC) domains. ACs catalyze the conversion of ATP to 3',5'-cyclic adenosine monophosphate (cAMP) while GCs catalyze the reaction of GTP to 3',5'-cyclic guanosine monophosphate (cGMP). Both cAMP and cGMP are now recognized as essential components of many plant responses, including responses to peptidic hormones. Here we describe the approach that led to the discovery of the Plant Natriuretic Peptide Receptor (PNP receptor), including a protocol for the identification of currently undiscovered peptidic interactions, and the subsequent application of computational methods for the identification of AC and/or GC domains in such interacting receptor candidates.

A Genome Sequence Assembly of the Phototactic and Optogenetic Model Fungus Blastocladiella emersonii Reveals a Diversified Nucleotide-Cyclase Repertoire.

The chytrid fungus Blastocladiella emersonii produces spores with swimming tails (zoospores); these cells can sense and swim toward light. Interest in this species stems from ongoing efforts to develop B. emersonii as a model for understanding the evolution of phototaxis and the molecular cell biology of the associated optogenetic circuits. Here, we report a highly contiguous genome assembly and gene annotation of the B. emersonii American Type Culture Collection 22665 strain. We integrate a PacBio long-read library with an Illumina paired-end genomic sequence survey leading to an assembly of 21 contigs totaling 34.27 Mb. Using these data, we assess the diversity of sensory system encoding genes. These analyses identify a rich complement of G-protein-coupled receptors, ion transporters, and nucleotide cyclases, all of which have been diversified by domain recombination and tandem duplication. In many cases, these domain combinations have led to the fusion of a protein domain to a transmembrane domain, tying a putative signaling function to the cell membrane. This pattern is consistent with the diversification of the B. emersonii sensory-signaling systems, which likely plays a varied role in the complex life cycle of this fungus.

Cyclic nucleotide-induced helical structure activates a TIR immune effector.

Cyclic nucleotide signalling is a key component of antiviral defence in all domains of life. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway1, which originated in bacteria2. These defence systems require a sensor domain to bind the cyclic nucleotide and are often coupled with an effector domain that, when activated, causes cell death by destroying essential biomolecules3. One example is the Toll/interleukin-1 receptor (TIR) domain, which degrades the essential cofactor NAD+ when activated in response to infection in plants and bacteria2,4,5 or during programmed nerve cell death6. Here we show that a bacterial antiviral defence system generates a cyclic tri-adenylate that binds to a TIR-SAVED effector, acting as the 'glue' to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organize and complete a composite active site, allowing NAD+ degradation. Activation requires extended filament formation, both in vitro and in vivo. Our study highlights an example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

EP115: Two novel NPR2 variants in a patient with acromesomelic dysplasia - Maroteaux type

Acromesomelic dysplasia, Maroteaux type (AMDM) is a rare autosomal recessive skeletal dysplasia characterized by severe disproportionate short stature (adult height < 120 cm), dolichocephaly, acro- and mesomelic shortening of the limbs, short, broad fingers and flat feet. Radiographic findings include bowed radius with a short ulna, dolichocephaly, platyspondyly and narrowing of the lumbar interpedicular distances. Intelligence is unaffected. The condition is typically diagnosed at birth or in infancy, but features become more apparent over time because of the loss of linear growth. AMDM is caused by biallelic loss of function variants in the NPR2 gene which encodes the natriuretic peptide receptor-B (NPR-B). The NPR2 gene encodes a transmembrane receptor that is critical in endochondral bone formation. Forty four percent of variants in NPR2 occur in the ligand binding domain, 8% in the transmembrane domain, 26% in the protein kinase domain and 21% in the nucleotide cyclase domain. A seven-month-old was seen with clinical findings suggestive of a skeletal dysplasia. She was born at 36 weeks following a pregnancy complicated by maternal polysubstance use and poor prenatal care. Clinical findings were shortening of all limb segments, trident hands, weak grasp, and overlapping second and third toes bilaterally. Radiographic findings were shortening of all the long bones with metaphyseal flaring, short and broad metatarsals, metacarpals and phalanges also with mild metaphyseal widening, platyspondyly with mild anterior vertebral body beaking in the upper lumbar spine. Cervical CT examination revealed platyspondyly of the suboccipital vertebrae and decreased AP diameter of the spinal canal at C1 secondary to anterior bowing of the posterior arch. A multi-gene skeletal dysplasia panel identified twelve variants, including two variants in NPR2. NPR2 c.1699G>T was classified as pathogenic, causing a premature termination at amino acid 567 in the kinase homology domain of the transmembrane protein. While classified as a VUS, the c.1123G>A missense variant was predicted to cause a substitution at amino acid 375 and falls at the last nucleotide of exon 4, which is part of the consensus splice site for this exon. The patient’s mother tested positive for the pathogenic variant and negative for the VUS in NPR2. Acromesomelic dysplasia, Maroteaux type is a rare skeletal dysplasia with limited published phenotypic and molecular information available. The described patient presented a diagnostic conundrum because only one of the variants had been characterized as pathogenic. The variant of uncertain significance is located in the ligand binding domain of the NPR2 gene, in which the majority of reported variants in this gene are found. In silico algorithms predict this variant to be deleterious to the protein structure and function but these predictions have not been confirmed by functional studies. Furthermore, missense variants within the consensus splice sites commonly cause abnormal splicing. This variant is not found in population databases. While paternal samples are not available to confirm that the variants are in trans, the patient was given the diagnosis of AMDM based on her clinical presentation. Molecular analysis and clinical description of other patients with AMDM will continue to characterize disease causing variants and natural history of this rare disease.

Elaborating the Functional Roles of a Leucine-Rich Repeat Protein from &amp;lt;i&amp;gt;Arabidopsis thaliana&amp;lt;/i&amp;gt;

Plants, just like any other living organism, naturally get attacked by various pathogenic microorganisms such as bacteria, fungi and viruses. However, unlike animals that utilize their specialized circulatory macrophage system to protect themselves, plants instead use a multi-layered complex system termed the plant innate immunity, which recognizes pathogens and transducing downstream defense responses. They have developed a unique type of trans-membrane receptors or R proteins, which extracellularly, are capable of recognizing pathogen-associated molecular patterns (PAMP) such as flagellin and chitin, while intracellularly, they activate their harbored nucleotide cyclases (NCs) such as adenylyl cyclases (ACs), to generate second messenger molecules such as 3’,5’-cyclic adenosine monophosphate (cAMP), which then propagates and magnifies the defense response. To date, only a single R protein from Arabidopsis thaliana (AtLRR) has been shown to possess AC activity as well as having the ability to defend plants against infection by biotrophic and hemi-biotrophic pathogens. Therefore, in order to further broaden information around the functional roles of this protein (AtLRR), we explored it further, using an array of web-based tools or bioinformatics. These included structural analysis, anatomical expression analysis, developmental expression analysis, co-expression analysis, functional enrichment analysis, stimulus-specific expression analysis and promoter analysis. Findings from structural analysis showed that AtLRR is a multi-domain, trans-membrane molecule that is multi-functional, and thus consistent with all known R-proteins. Findings from anatomical and developmental expression analyses showed that AtLRR is mostly expressed in pollen grains and flowers, senescing leaves as well as during the development of seeds, shoots, roots, seedlings, leaves, flowers, and siliques, linking it to the three key plant physiological processes of reproduction, defense and development respectively. Lastly, findings from co-expression, functional enrichment, stimulus-specific expression and promoter analyses, showed that AtLRR is mostly co-expressed with several other proteins linked to disease resistance, plant reproduction and plant development. Activities and functions of such protein are also commonly regulated by cAMP via a common W-box promoter. So, all in all, our study managed to establish that besides being strongly involved in disease resistance against biotrophic and hemi-biotrophic pathogens, AtLRR also plays key roles in plant development (seed, shoot, root, seedling, leaf, and silique development) and reproduction (flowering, and pollen tube growth and re-orientation), whereby it effects its functions via a W-box or WRKY transcription factor, TTGACY, mediated by cAMP.

Unusual features and localization of the membrane kinome of Trypanosoma brucei.

In many eukaryotes, multiple protein kinases are situated in the plasma membrane where they respond to extracellular ligands. Ligand binding elicits a signal that is transmitted across the membrane, leading to activation of the cytosolic kinase domain. Humans have over 100 receptor protein kinases. In contrast, our search of the Trypanosoma brucei kinome showed that there were only ten protein kinases with predicted transmembrane domains, and unlike other eukaryotic transmembrane kinases, seven are predicted to bear multiple transmembrane domains. Most of the ten kinases, including their transmembrane domains, are conserved in both Trypanosoma cruzi and Leishmania species. Several possess accessory domains, such as Kelch, nucleotide cyclase, and forkhead-associated domains. Surprisingly, two contain multiple regions with predicted structural similarity to domains in bacterial signaling proteins. A few of the protein kinases have previously been localized to subcellular structures such as endosomes or lipid bodies. We examined the localization of epitope-tagged versions of seven of the predicted transmembrane kinases in T. brucei bloodstream forms and show that five localized to the endoplasmic reticulum. The last two kinases are enzymatically active, integral membrane proteins associated with the flagellum, flagellar pocket, or adjacent structures as shown by both fluorescence and immunoelectron microscopy. Thus, these kinases are positioned in structures suggesting participation in signal transduction from the external environment.

CdgL is a degenerate nucleotide cyclase domain protein affecting flagellin synthesis and motility in Bacillus thuringiensis

In Bacillus subtilis, motility genes are expressed in a hierarchical pattern – governed by the σD transcription factor and other proteins such as the EpsE molecular clutch and SlrA/SlrR regulator proteins. In contrast, motile species in the Bacillus cereus group seem to express their motility genes in a non-hierarchical pattern, and less is known about their regulation, also given that no orthologs to σD, EpsE, SlrA or SlrR are found in B. cereus group genomes. Here we show that deletion of cdgL (BTB_RS26690/BTB_c54300) in Bacillus thuringiensis 407 (cry-) resulted in a six-to ten-fold downregulation of the entire motility locus, and loss of flagellar structures and swimming motility. cdgL is unique to the B. cereus group and is found in all phylogenetic clusters in the population except for group I, which comprises isolates of non-motile Bacillus pseudomycoides. Analysis of RNA-Seq data revealed cdgL to be expressed in a three-gene operon with a NupC like nucleoside transporter, and a putative glycosyl transferase for which transposon-based gene inactivation was previously shown to produce a similar phenotype to cdgL deletion. Interestingly, all three proteins were predicted to be membrane-bound and may provide a concerted function in the regulation of B. cereus group motility.

Advances, Perspectives and Potential Engineering Strategies of Light-Gated Phosphodiesterases for Optogenetic Applications.

The second messengers, cyclic adenosine 3′-5′-monophosphate (cAMP) and cyclic guanosine 3′-5′-monophosphate (cGMP), play important roles in many animal cells by regulating intracellular signaling pathways and modulating cell physiology. Environmental cues like temperature, light, and chemical compounds can stimulate cell surface receptors and trigger the generation of second messengers and the following regulations. The spread of cAMP and cGMP is further shaped by cyclic nucleotide phosphodiesterases (PDEs) for orchestration of intracellular microdomain signaling. However, localized intracellular cAMP and cGMP signaling requires further investigation. Optogenetic manipulation of cAMP and cGMP offers new opportunities for spatio-temporally precise study of their signaling mechanism. Light-gated nucleotide cyclases are well developed and applied for cAMP/cGMP manipulation. Recently discovered rhodopsin phosphodiesterase genes from protists established a new and direct biological connection between light and PDEs. Light-regulated PDEs are under development, and of demand to complete the toolkit for cAMP/cGMP manipulation. In this review, we summarize the state of the art, pros and cons of artificial and natural light-regulated PDEs, and discuss potential new strategies of developing light-gated PDEs for optogenetic manipulation.

Tlr0485 is a cAMP-activated c-di-GMP phosphodiesterase in a cyanobacterium Thermosynechococcus.

Second messenger molecules are crucial components of environmental signaling systems to integrate multiple inputs and elicit physiological responses. Among various kinds of second messengers, cyclic nucleotides cAMP and cyclic di-GMP (c-di-GMP) play pivotal roles in bacterial environmental responses. However, how these signaling systems are interconnected for a concerted regulation of cellular physiology remains elusive. In a thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN, incident light color is sensed by cyanobacteriochrome photoreceptors to transduce the light information to the levels of c-di-GMP, which induces cellular aggregation probably via cellulose synthase activation. Herein, we identified that Tlr0485, which is composed of a cGMP-specific phosphodiesterases, adenylate cyclases, and FhlA (GAF) domain and an HD-GYP domain, is a cAMP-activated c-di-GMP phosphodiesterase. We also show biochemical evidence that the two class-III nucleotide cyclases, Cya1 and Cya2, are both adenylate cyclases to produce cAMP in T. vulcanus. The prevalence of cAMP-activated c-di-GMP phosphodiesterase genes in cyanobacterial genomes suggests that the direct crosstalk between cAMP and c-di-GMP signaling systems may be crucial for cyanobacterial environmental responses.

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q0 or Q0 H2 ). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q0 and O2 , or NADH and Q0 H2 respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.

Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis

cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fine-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.

Bioinformatic analysis of nucleotide cyclase functional centers and development of ACpred webserver

Cyclic mononucleotides, in particular 3',5'-cyclic guanosine monophosphate (cGMP) and 3',5'-cyclic adenosine monophosphate (cAMP), are molecular signals that mediate a myriad of biological responses in organisms across the tree of life. In plants, they transduce signals such as hormones and peptides perceived at receptors on the cell surface into the cytoplasm to orchestrate a cascade of biochemical reactions that enable them to grow and develop, and adapt to light, hormones, salt and drought stresses as well as pathogens. However, their generating enzymes (guanylyl cyclases, GCs and adenylyl cyclases, ACs) have just been recently discovered and are still poorly understood. Here, we employed a computational approach to probe the physicochemical properties of the catalytic centers of these enzymes and the knowledge of which, was used to create a web-based tool, ACPred (http://gcpred.com/acpred) for the prediction of AC functional centers from amino acid sequence. Understanding the nature of such catalytic centers have enabled the creation of predictive tools such as ACPred which will in turn, facilitate the discovery of novel cellular components across different systems.

Discovery of Novel Functional Centers With Rationally Designed Amino Acid Motifs

Plants are constantly exposed to environmental stresses and in part due to their sessile nature, they have evolved signal perception and adaptive strategies that are distinct from those of other eukaryotes. This is reflected at the cellular level where receptors and signalling molecules cannot be identified using standard homology-based searches querying with proteins from prokaryotes and other eukaryotes. One of the reasons for this is the complex domain architecture of receptor molecules. In order to discover hidden plant signalling molecules, we have developed a motif-based approach designed specifically for the identification of functional centers in plant molecules. This has made possible the discovery of novel components involved in signalling and stimulus-response pathways; the molecules include cyclic nucleotide cyclases, a nitric oxide sensor and a novel target for the hormone abscisic acid. Here, we describe the major steps of the method and illustrate it with recent and experimentally confirmed molecules as examples. We foresee that carefully curated search motifs supported by structural and bioinformatic assessments will uncover many more structural and functional aspects, particularly of signalling molecules.

Discovery of Oligonucleotide Signaling Mediated by CRISPR-Associated Polymerases Solves Two Puzzles but Leaves an Enigma.

The signature component of type III CRISPR-Cas systems is the Cas10 protein that consists of two Palm domains homologous to those of DNA and RNA polymerases and nucleotide cyclases and an HD nuclease domain. However, until very recently, the activity of the Palm domains and their role in CRISPR function have not been experimentally established. Most of the type III CRISPR-Cas systems and some type I systems also encompass proteins containing the CARF (CRISPR-associated Rossmann fold) domain that has been predicted to regulate CRISPR functions via nucleotide binding, but its function in CRISPR-Cas remained obscure. Two independent recent studies show that the Palm domain of Cas10 catalyzes synthesis of oligoadenylates, which bind the CARF domain of the Csm6 protein and activate its RNase domain that cleaves foreign transcripts enabling interference by type III CRISPR-Cas. In one coup, these findings resolved two long-standing puzzles of CRISPR biology and reveal a new regulatory pathway that governs the CRISPR response. However, the full extent of this pathway, and especially the driving forces behind the evolution of this complex mechanism of CRISPR-Cas activation, remains to be uncovered.

Die Kontrolle zyklischer Nukleotide mittels Licht

Sensory photoreceptors underpin diverse adaptive responses in nature and serve as genetically encoded, light-gated actuators in optogenetics. Photoactivated nucleotide cyclases (PACs) enable light-dependent control over the formation of cyclic nucleotides (cNMPs). Conversely, the engineered, red-light-activated phosphodiesterase LAPD mediates the hydrolytic breakdown of cNMPs. In combination, PACs and LAPD hence unlock precise and graded control over intracellular cNMPs and associated processes.

Identification of bacterial guanylate cyclases.

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRPG, constructed here. While wild-type CRP is activated exclusively by cAMP,_CRPG can be activated by either cAMP or cGMP. Using CRP- and CRPG-dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties.

Computational recognition and analysis of hitherto uncharacterized nucleotide cyclase-like proteins in bacteria.

Evolutionary relationship between class III nucleotide cyclases and an uncharacterized set of bacterial proteins from Actinobacteria, Bacteroidetes and Proteobacteria has been recognized and analyzed. Detailed analyses of sequence and structural features resulted in the recognition of potential cyclase function conferring residues and presence of signature topological motif (βααββαβ) in the uncharacterized set of bacterial proteins. Lack of transmembrane domains and signal peptide cleavage sites is suggestive of their cytosolic subcellular localization. Furthermore, analysis on evolutionarily conserved gene clusters of the predicted nucleotide cyclase-like proteins and their evolutionary relationship with nucleotide cyclases suggest their participation in cellular signalling events. Our analyses suggest expansion of class III nucleotide cyclases.ReviewersThis article was reviewed by Eugene Koonin and Michael Gromiha.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0130-9) contains supplementary material, which is available to authorized users.

Common Structural Core of Three-Dozen Residues Reveals Intersuperfamily Relationships.

Identification of relationships among protein families or superfamilies is a challenge. However, functionally essential protein regions typically retain structural integrity, even when the corresponding protein sequences evolve. Consequently, comparison of protein structures enables deeper phylogenetic analyses than achievable through the use of sequence information only. Here, we focus on a group of distantly related viral and cellular enzymes involved in nucleic acid or nucleotide processing and synthesis. All these enzymes share an apparently similar protein fold at their active site, which resembles the palm subdomain of the right-hand-shaped polymerases. Using a structure-based hierarchical clustering method, we identified a common structural core of 36 equivalent residues for this functionally diverse group of enzymes, representing five protein superfamilies. Based on the properties of these 36 residues, we deduced a structural distance-based tree in which the proteins were accurately clustered according to the established family classification. Within this tree, the enzymes catalyzing genomic nucleic acid replication or transcription were separated from those performing supplementary nucleic acid or nucleotide processing functions. In addition, we found that the family Y DNA polymerases are structurally more closely related to the nucleotide cyclase superfamily members than to the other members of the DNA/RNA polymerase superfamily, and these enzymes share 88 equivalent residues comprising a Β: 1- Α: 1- Α: 2- Β: 2- Β: 3- Α: 3- Β: 4- Α: 4- Β: 5 fold. The results highlight the power of structure-based hierarchical clustering in identifying remote evolutionary relationships. Furthermore, our study implies that a protein substructure of only three-dozen residues can contain a substantial amount of information on the evolutionary history of proteins.