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PubMed1998

Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila.

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and Mössbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.

Amino Acid Sequence↗
PubMed1999

A comparison of eubacterial and archaeal structure-specific 5'-exonucleases.

The 5'-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structure-specific 5'-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5' strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase. In this paper, we define the substrate requirements for the 5'-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum. The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity.

Archaea↗
PubMed1999

An archaebacterial ATPase, homologous to ATPases in the eukaryotic 26 S proteasome, activates protein breakdown by 20 S proteasomes.

In eukaryotes, the 20 S proteasome is the proteolytic core of the 26 S proteasome, which degrades ubiquitinated proteins in an ATP-dependent process. Archaebacteria lack ubiquitin and 26 S proteasomes but do contain 20 S proteasomes. Many archaebacteria, such as Methanococcus jannaschii, also contain a gene (S4) that is highly homologous to the six ATPases in the 19 S (PA700) component of the eukaryotic 26 S proteasome. To test if this putative ATPase may regulate proteasome function, we expressed it in Escherichia coli and purified the 50-kDa product as a 650-kDa complex with ATPase activity. When mixed with the well characterized 20 S proteasomes from Thermoplasma acidophilum and ATP, this complex stimulated degradation of several unfolded proteins 8-25-fold. It also stimulated proteolysis by 20 S proteasomes from another archaebacterium and mammals. This effect required ATP hydrolysis since ADP and the nonhydrolyzable analog, 5'-adenylyl beta, gamma-imidophosphate, were ineffective. CTP and to a lesser extent GTP and UTP were also hydrolyzed and also stimulated proteolysis. We therefore named this complex PAN for proteasome-activating nucleotidase. However, PAN did not promote the degradation of small peptides, which, unlike proteins, should readily diffuse into the proteasome. This ATPase complex appears to have been the evolutionary precursor of the eukaryotic 19 S complex, before the coupling of proteasome function to ubiquitination.

Adenosine Triphosphatases↗
PubMed2000

Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system.

Cation hexaammines and related compounds are chemically stable analogs of the hydrated form of cations, particularly Mg(2+). We tested the ability of several of these compounds to inhibit transport by the CorA or MgtB Mg(2+) transport systems or the PhoQ receptor kinase for Mg(2+) in Salmonella typhimurium. Cobalt(III)-, ruthenium(II)-, and ruthenium(III)-hexaammines were potent inhibitors of CorA-mediated influx. Cobalt(III)- and ruthenium(III)chloropentaammines were slightly less potent inhibitors of CorA. The compounds inhibited uptake by the bacterial S. typhimurium CorA and by the archaeal Methanococcus jannaschii CorA, which bear only 12% identity in the extracellular periplasmic domain. Cation hexaammines also inhibited growth of S. typhimurium strains dependent on CorA for Mg(2+) uptake but not of isogenic strains carrying a second Mg(2+) uptake system. In contrast, hexacyano-cobaltate(III) and ruthenate(II)- and nickel(II)hexaammine had little effect on uptake. The inhibition by the cation hexaammines was selective for CorA because none of the compounds had any effect on transport by the MgtB P-type ATPase Mg(2+) transporter or the PhoQ Mg(2+) receptor kinase. These results demonstrate that cation hexaammines are potent and highly selective inhibitors of the CorA Mg(2+) transport system and further indicate that the initial interaction of the CorA transporter is with a fully hydrated Mg(2+) cation.

Amines↗
PubMed2001

A novel quaternary structure of the dimeric alpha-crystallin domain with chaperone-like activity.

alphaB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human alphaB-crystallin (alphaB57-157), a dimeric protein that comprises the alpha-crystallin domain of the alphaB-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the alpha-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the alpha-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the alpha-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite beta-sheet. This model agrees with the recent spin labeling results and suggests that the alphaB-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of alphaB-crystallin complexes of variable sizes and compositions.

Amino Acid Sequence↗
PubMed2001

Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination.

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.

Amino Acid Sequence↗
PubMed2002

The structure of a binary complex between a mammalian mevalonate kinase and ATP: insights into the reaction mechanism and human inherited disease.

Mevalonate kinase catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the pathways of isoprenoids and sterols. Deficiency in mevalonate kinase activity has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). The crystal structure of rat mevalonate kinase in complex with MgATP has been determined at 2.4-A resolution. Each monomer of this dimeric protein is composed of two domains with its active site located at the domain interface. The enzyme-bound ATP adopts an anti conformation, in contrast to the syn conformation reported for Methanococcus jannaschii homoserine kinase. The Mg(2+) ion is coordinated to both beta- and gamma-phosphates of ATP and side chains of Glu(193) and Ser(146). Asp(204) is making a salt bridge with Lys(13), which in turn interacts with the gamma-phosphate. A model of mevalonic acid can be placed near the gamma-phosphoryl group of ATP; thus, the C5 hydroxyl is located within 4 A from Asp(204), Lys(13), and the gamma-phosphoryl of ATP. This arrangement of residues strongly suggests: 1) Asp(204) abstracts the proton from C5 hydroxyl of mevalonate; 2) the penta-coordinated gamma-phosphoryl group may be stabilized by Mg(2+), Lys(13), and Glu(193); and 3) Lys(13) is likely to influence the pK(a) of the C5 hydroxyl of the substrate. V377I and I268T are the most common mutations found in patients with HIDS. Val(377) is located over 18 A away from the active site and a conservative replacement with Ile is unlikely to yield an inactive or unstable protein. Ile-268 is located at the dimer interface, and its Thr substitution may disrupt dimer formation.

Adenosine Triphosphate↗
PubMed2002

A novel candidate for the true fructose-1,6-bisphosphatase in archaea.

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes of the gluconeogenic pathway. Although enzyme activity had been detected in Archaea, the corresponding gene had not been identified until a presumable inositol monophosphatase gene from Methanococcus jannaschii was found to encode a protein with both inositol monophosphatase and FBPase activities. Here we display that a gene from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, which does not correspond to the inositol monophosphatase gene from M. jannaschii, displays high FBPase activity. The FBPase from strain KOD1 was partially purified, its N-terminal amino acid sequence was determined, and the gene (Tk-fbp) was cloned. Tk-fbp encoded a protein of 375 amino acid residues with a molecular mass of 41,658 Da. The recombinant Tk-Fbp was purified and characterized. Tk-Fbp catalyzed the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate following Michaelis-Menten kinetics with a K(m) value of 100 microm toward fructose 1,6-bisphosphate, and a k(cat) value of 17 s(-1) subunit(-1) at 95 degrees C. Unlike the inositol monophosphatase from M. jannaschii, Tk-Fbp displayed strict substrate specificity for fructose 1,6-bisphosphate. Activity was enhanced by Mg(2+) and dithioerythritol, and was slightly inhibited by fructose 2,6-bisphosphate. AMP did not inhibit the enzyme activity. We examined whether expression of Tk-fbp was regulated at the transcription level. High levels of Tk-fbp transcripts were detected in cells grown on pyruvate or amino acids, whereas no transcription was detected when starch was present in the medium. Orthologue genes corresponding to Tk-fbp with high similarity are present in all the complete genome sequences of thermophilic Archaea, including M. jannaschii, Pyrococcus furiosus, Sulfolobus solfataricus, and Archaeoglobus fulgidus, but are yet to be assigned any function. Taking into account the high FBPase activity of the protein, the strict substrate specificity, and its sugar-repressed gene expression, we propose that Tk-Fbp may represent the bona fide FBPase in Archaea.

Amino Acid Sequence↗
PubMed2002

Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine.

Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and Söll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.

Acylation↗
PubMed2004

A dual role for the N-terminal region of Mycobacterium tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins.

The N-terminal regions, which are highly variable in small heat-shock proteins, were found to be structurally disordered in all the 24 subunits of Methanococcus jannaschii Hsp16.5 oligomer and half of the 12 subunits of wheat Hsp16.9 oligomer. The structural and functional roles of the corresponding region (potentially disordered) in Mycobacterium tuberculosis Hsp16.3, existing as nonamers, were investigated in this work. The data demonstrate that the mutant Hsp16.3 protein with 35 N-terminal residues removed (DeltaN35) existed as trimers/dimers rather than as nonamers, failing to bind the hydrophobic probe (1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid) and exhibiting no chaperone-like activity. Nevertheless, another mutant protein with the C-terminal extension (of nine residues) removed, although existing predominantly as dimers, exhibited efficient chaperone-like activity even at room temperatures, indicating that pre-existence as nonamers is not a prerequisite for its chaperone-like activity. Meanwhile, the mutant protein with both the N- and C-terminal ends removed fully exists as a dimer lacking any chaperone-like activity. Furthermore, the N-terminal region alone, either as a synthesized peptide or in fusion protein with glutathione S-transferase, was capable of interacting with denaturing proteins. These observations strongly suggest that the N-terminal region of Hsp16.3 is not only involved in self-oligomerization but also contains the critical site for substrate binding. Such a dual role for the N-terminal region would provide an effective mechanism for the small heat-shock protein to modulate its chaperone-like activity through oligomeric dissociation/reassociation. In addition, this study demonstrated that the wild-type protein was able to form heterononamers with DeltaN35 via subunit exchange at a subunit ratio of 2:1. This implies that the 35 N-terminal residues in three of the nine subunits in the wild-type nonamer are not needed for the assembly of nonamers from trimers and are thus probably structurally disordered.

Amino Acid Sequence↗
PubMed2005

Exosite modules guide substrate recognition in the ZiPD/ElaC protein family.

Escherichia coli ZiPD is the best characterized protein encoded by the elaC gene family and is a model for the 3'-pre-tRNA processing endoribonucleases (tRNase Z). A metal ligand-based sequence alignment of ZiPD with metallo-beta-lactamase domain proteins of known crystallographic structure identifies a ZiPD-specific sequence insertion of approximately 50 residues, which we will refer to as the ZiPD exosite. Functionally characterized ZiPD homologs from Bacillus subtilis, Methanococcus janaschii, and human share the presence of the ZiPD exosite, which is also present in the amino-terminal, but not in the carboxyl-terminal, domain of ElaC2 proteins. Another class of functionally characterized tRNase Z enzymes from Thermotoga maritima and Arabidopsis thaliana lack characteristic motifs in the exosite but possess a sequence segment with clustered basic amino acid residues. As an experimental attempt to investigate the function of the exosite we constructed a ZiPD variant that lacks this module (ZiPDDelta). ZiPDDelta has almost wild-type-like catalytic properties for hydrolysis of the small, chromogenic substrate bis(p-nitrophenyl) phosphate. Removal of the ZiPD exosite only affects k(cat), which is reduced by less than 40%, whereas both K' andthe Hill coefficient (measures of the substrate affinity and cooperativity, respectively) remain unchanged. Hence, the exosite is not required for the intrinsic phosphodiesterase activity of ZiPD. Removal of the exosite also does not affect the dimerization properties of ZiPD. In contrast to the wild-type enzyme, ZiPDDelta does not process pre-tRNA, and gel shift assays demonstrate that only the wild-type enzyme, but not ZiPDDelta, binds mature tRNA. These findings show that the exosite is essential for pre-tRNA recognition. In conclusion, we identify a ZiPD exosite that guides physiological substrate recognition in the ZiPD/ElaC protein family.

Amino Acid Motifs↗
PubMed2005

Biosynthesis of vitamin B2: diastereomeric reaction intermediates of archaeal and non-archaeal riboflavin synthases.

The dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase affords riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. A pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazines has been identified earlier as a catalytically competent reaction intermediate of the Escherichia coli enzyme. Acid quenching of reaction mixtures of riboflavin synthase of Methanococcus jannaschii, a paralog of 6,7-dimethyl-8-ribityllumazine synthase devoid of similarity with riboflavin synthases of eubacteria and eukaryotes, afforded a compound whose optical absorption and NMR spectra resemble that of the pentacyclic E. coli riboflavin synthase intermediate, whereas the circular dichroism spectra of the two compounds have similar envelopes but opposite signs. Each of the compounds could serve as a catalytically competent intermediate for the enzyme by which it was produced, but not vice versa. All available data indicate that the respective pentacyclic intermediates of the M. jannaschii and E. coli enzymes are diastereomers.

Archaea↗
PubMed2006

Salt tolerance of archaeal extremely halophilic lipid membranes.

The membranes of extremely halophilic Archaea are characterized by the abundance of a diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me), which accounts for 50-80 mol% of the polar lipids, and by the absence of phospholipids with choline, ethanolamine, inositol, and serine head groups. These membranes are stable in concentrated 3-5 m NaCl solutions, whereas membranes of non-halophilic Archaea, which do not contain PGP-Me, are unstable and leaky under such conditions. By x-ray diffraction and vesicle permeability measurements, we demonstrate that PGP-Me contributes in an essential way to membrane stability in hypersaline environments. Large unilamellar vesicles (LUV) prepared from the polar lipids of extreme halophiles, Halobacterium halobium and Halobacterium salinarum, retain entrapped carboxyfluorescein and resist aggregation in the whole range 0-4 m NaCl, similarly to LUV prepared from purified PGP-Me. By contrast, LUV made of polar lipid extracts from moderately halophilic and non-halophilic Archaea (Methanococcus jannaschii, Methanosarcina mazei, Methanobrevibacter smithii) are leaky and aggregate at high salt concentrations. However, adding PGP-Me to M. mazei lipids results in gradual enhancement of LUV stability, correlating with the PGP-Me content. The LUV data are substantiated by the x-ray results, which show that H. halobium and M. mazei lipids have dissimilar phase behavior and form different structures at high NaCl concentrations. H. halobium lipids maintain an expanded lamellar structure with spacing of 8.5-9 nm, which is stable up to at least 100 degrees C in 2 m NaCl and up to approximately 60 degrees C in 4 m NaCl. However, M. mazei lipids form non-lamellar structures, represented by the Pn3m cubic phase and the inverted hexagonal H(II) phase. From these data, the forces preventing membrane aggregation in halophilic Archaea appear to be steric repulsion, because of the large head group of PGP-Me, or possibly out-of-plane bilayer undulations, rather than electrostatic repulsion attributed to the doubly charged PGP-Me head group.

Archaeal Proteins↗
PubMed2005

Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY.

During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation.

Amino Acid Substitution↗
PubMed2002

Identification of a novel family of sequence repeats among prokaryotes.

The rapid increase in genomic sequences provides new opportunities for comparative genomics. In this report, we describe a novel family of repeat sequences that is present in Bacteria and Archaea but not in Eukarya. The repeat loci typically consisted of repetitive stretches of nucleotides with a length of 25 to 37 bp alternated by nonrepetitive DNA spacers of approximately equal size as the repeats. The nucleotide sequences and the size of the repeats were highly conserved within a species, but between species the sequences showed no similarity. Due to their characteristic structure, we have designated this family of repeat loci as SPacers Interspersed Direct Repeats (SPIDR). The SPIDR loci were identified in more than forty different prokaryotic species. Individual species such as Mycobacterium tuberculosis contain one SPIDR locus, while other species such as Methanococcus jannaschii contained up to 20 different loci. The number of repeats in a locus varies greatly from two repeats to several dozens of repeats. The SPIDR loci were flanked by a common 300-500-bp leader sequence, which appeared to be conserved within a species but not between species. The SPIDR locus of M. tuberculosis is extensively used for strain typing. The finding of SPIDR loci in other prokaryotes, including the pathogens Salmonella, Campylobacter, and Pasteurella may extend this surveillance to other species.

Archaea↗
PubMed1997

Statistical modeling, phylogenetic analysis and structure prediction of a protein splicing domain common to inteins and hedgehog proteins.

Inteins, introns spliced at the protein level, and the hedgehog family of proteins involved in eucaryotic development both undergo autocatalytic proteolysis. Here, a specific and sensitive hidden Markov model (HMM) of protein splicing domain shared by inteins and the hedgehog proteins has been trained and employed for further analysis. The HMM characterizes the common features of this domain including the position where a site-specific DNA endonuclease domain is inserted in the majority of the inteins. The HMM was used to identify several new putative inteins, such as that in the Methanococcus jannaschii klbA protein, and to generate a multiple sequence alignment of sequences possessing this domain. Phylogenetic analysis suggests that hedgehog proteins evolved from inteins. Secondary and tertiary structure predictions suggest that the domain has a structure similar to a beta-sandwich. Similarities between the serine protease cleavage mechanism and the protein splicing reaction mechanism are discussed. Examination of the locations of inteins indicates that they are not inserted randomly in an extein, but are often inserted at functionally important positions in the host proteins. A specific and sensitive HMM for a domain present in klbA proteins identified several additional bacterial and archaeal family members, and analysis of the site of insertion of the intein suggests residues that may be functionally important. This domain may play a role in formation of surface-associated protein complexes.

Algorithms↗
PubMed1998

Statistical modelling and phylogenetic analysis of a deaminase domain.

Deamination reactions are catalyzed by a variety of enzymes including those involved in nucleoside/nucleotide metabolism and cytosine to uracil (C-->U) and adenosine to inosine (A-->I) mRNA editing. The active site of the deaminase (DM) domain in these enzymes contains a conserved histidine (or rarely cysteine), two cysteines and a glutamate proposed to act as a proton shuttle during deamination. Here, a statistical model, a hidden Markov model (HMM), of the DM domain has been created which identifies currently known DM domains and suggests new DM domains in viral, bacterial and eucaryotic proteins. However, no DM domains were identified in the currently predicted proteins from the archaeon Methanococcus jannaschii and possible causes for, and a potential means to ameliorate this situation are discussed. In some of the newly identified DM domains, the glutamate is changed to a residue that could not function as a proton shuttle and in one instance (Mus musculus spermatid protein TENR) the cysteines are also changed to lysine and serine. These may be non-competent DM domains able to bind but not act upon their substrate. Phylogenetic analysis using an HMM-generated alignment of DM domains reveals three branches with clear substructure in each branch. The results suggest DM domains that are candidates for yeast, platyhelminth, plant and mammalian C-->U and A-->I mRNA editing enzymes. Some bacterial and eucaryotic DM domains form distinct branches in the phylogenetic tree suggesting the existence of common, novel substrates.

Amino Acid Sequence↗
PubMed1999

A comparison of sequence and structure protein domain families as a basis for structural genomics.

MOTIVATION: Protein families can be defined based on structure or sequence similarity. We wanted to compare two protein family databases, one based on structural and one on sequence similarity, to investigate to what extent they overlap, the similarity in definition of corresponding families, and to create a list of large protein families with unknown structure as a resource for structural genomics. We also wanted to increase the sensitivity of fold assignment by exploiting protein family HMMs. RESULTS: We compared Pfam, a protein family database based on sequence similarity, to Scop, which is based on structural similarity. We found that 70% of the Scop families exist in Pfam while 57% of the Pfam families exist in Scop. Most families that occur in both databases correspond well to each other, but in some cases they are different. Such cases highlight situations in which structure and sequence approaches differ significantly. The comparison enabled us to compile a list of the largest families that do not occur in Scop; these are suitable targets for structure prediction and determination, and may be useful to guide projects in structural genomics. It can be noted that 13 out of the 20 largest protein families without a known structure are likely transmembrane proteins. We also exploited Pfam to increase the sensitivity of detecting homologs of proteins with known structure, by comparing query sequences to Pfam HMMs that correspond to Scop families. For SWISSPROT+TREMBL, this yielded an increase in fold assignment from 31% to 42% compared to using FASTA only. This method assigned a structure to 22% of the proteins in Saccharomyces cerevisiae, 24% in Escherichia coli, and 16% in Methanococcus jannaschii.

Computational Biology↗