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PubMed2000

Sequence and phylogenetic analysis of squid myosin-V: a vesicle motor in nerve cells.

We have shown that vesicles in the axoplasm of the squid giant axon move on actin filaments and that movement is inhibited by myosin V-specific antibodies [Tabb et al., 1998]. In the study reported in this article, experiments were performed to clone and sequence the cDNA for squid brain myosin V. Five proteolytic fragments of purified squid brain myosin V were analyzed by direct protein sequencing [Tabb et al., 1998]. Based on this sequence information, degenerate primers were constructed and used to isolate cDNA clones by PCR. Five clones, representing overlapping segments of the gene, were sequenced. The sequence data and the previous biochemical characterization of the molecule support the classification of this vesicle-associated myosin as a member of the class V myosins. Motif analysis of the head, neck, and tail domains revealed that squid MyoV has consensus sequences for all the motifs found in vertebrate members of the myosin V family of motor proteins. A phylogenetic tree was constructed from a sequence alignment by the neighbor-joining method, using Megalign (DNAStar, Madison, WI); the resulting phylogenetic tree showed that squid MyoV is more closely related to vertebrate MyoV (mouse dilute, chicken dilute, rat myr6, and human myo5a) than Drosophila and yeast (myo2, and myo4) myosins V. These new data on the phylogenetic relationships of squid myosin V to vertebrate myosin V strengthens the argument that myosin V functions as a vesicle motor in vertebrate neurons.

Amino Acid Motifs↗

PubMed2003

Steady-state kinetics and molecular evolution of Escherichia coli MenD [(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase], an anomalous thiamin diphosphate-dependent decarboxylase-carboligase.

(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide. The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others. However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate. Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction. The protein is a dimer and shows cooperativity with respect to both substrates. The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD. MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp. NRC-1 and the eukaryote Arabidopsis thaliana. Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP. A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis. There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.

Amino Acid Sequence↗

PubMed2005

Histidine at residue 99 and the transmembrane region of the precursor membrane prM protein are important for the prM-E heterodimeric complex formation of Japanese encephalitis virus.

The formation of the flavivirus prM-E complex is an important step for the biogenesis of immature virions, which is followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. In this study, the intracellular formation of the prM-E complex of Japanese encephalitis virus was investigated by baculovirus coexpression of prM and E in trans in Sf9 insect cells as analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of carboxyl-terminally truncated prM mutant baculoviruses was constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex by approximately 40% for the TM1 (at residues 130 to 147 [prM130-147]) truncation and 20% for TM2 (at prM153-167) truncation. Alanine-scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM binding element for stable prM-E heterodimeric complex formation. The single amino acid mutation at the His99 residue of prM abolishing the prM-E interaction was not due to reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse-chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus coexpression of prM and E proteins but not with the prM H99A mutant. Sequence alignment analysis was further conducted with different groups of flaviviruses to show that the prM H99 residues are generally conserved. Our findings are the first report to characterize the minimum binding elements of the prM protein that are involved in prM-E interactions of flaviviruses. This information, concerning a molecular framework for the prM protein, is considered to elucidate the structure/function relationship of the prM-E complex synthesis and provide the proper trajectory for flavivirus assembly and maturation.

Amino Acid Sequence↗

PubMed1999

Structure-function analysis of human cytochrome P-450 2B6 using a novel substrate, site-directed mutagenesis, and molecular modeling.

The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.

Animals↗

PubMed1995

The crystal structure of murine leukemia inhibitory factor.

We have determined the structure of murine leukemia inhibitory factor (LIF) by X-ray crystallography at 2.0 A resolution. The current crystal structure comprises native LIF residues 9 to 180 with 40 ordered water molecules. For this model the R value (with a bulk solvent correction) is 18.6% on all data from 20.0 A to 2.0 A with stereochemistry typified by root mean square deviations from ideal bond lengths of 0.015 A. The mainchain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony stimulating factor and growth hormone. Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved structural and surface character. Analysis of published mutagenesis data implicate two regions of receptor interaction which are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the growth hormone ligand/receptor complex requires additional, novel features to account for these data.

Amino Acid Sequence↗

PubMed2002

First molecular characterization of an unequal homologous alu-mediated recombination event responsible for hemophilia.

The large number of Alu repeats in the human genome provides abundant opportunities for unequal homologous recombination events that are responsible of several human diseases. We here describe a novel large FVIII gene deletion from a severe hemophilia A patient in which Alu-repetitive elements are directly involved in the origin of the mutation. Using a long-fragment PCR method, a approximately 23 kb deletion was delimited between introns 24 and 25. The resulting FVIII gene had a hybrid 2317-bp intron and lacked exon 25. Absence of exon 25 was confirmed at the RNA level. Multiple sequence alignment of this hybrid intron and normal introns 24 and 25 provided evidence of an homologous recombination event between two Alu repeats and the exact breakpoints were delimited to a 16 bp region. To our knowledge, this is the first report of hemophilia caused by unequal homologous Alu/Alu recombination. This mechanism, commonly related to genetic human disorders, may be involved in a significant number of hemophilia cases considering that FVIII is coded by an Alu-rich gene.

Alu Elements↗

PubMed2005

Crystal structure of human guanosine monophosphate reductase 2 (GMPR2) in complex with GMP.

Guanosine monophosphate reductase (GMPR) catalyzes the irreversible and NADPH-dependent reductive deamination of GMP to IMP, and plays a critical role in re-utilization of free intracellular bases and purine nucleosides. Here, we report the first crystal structure of human GMP reductase 2 (hGMPR2) in complex with GMP at 3.0 A resolution. The protein forms a tetramer composed of subunits adopting the ubiquitous (alpha/beta)8 barrel fold. Interestingly, the substrate GMP is bound to hGMPR2 through interactions with Met269, Ser270, Arg286, Ser288, and Gly290; this makes the conformation of the adjacent flexible binding region (residues 268-289) fixed, much like a door on a hinge. Structure comparison and sequence alignment analyses show that the conformation of the active site loop (residues 179-187) is similar to those of hGMPR1 and inosine monophosphate dehydrogenases (IMPDHs). We propose that Cys186 is the potential active site, and that the conformation of the loop (residues 129-133) suggests a preference for the coenzyme NADPH over NADH. This structure provides important information towards understanding the functions of members of the GMPR family.

Amino Acid Sequence↗

PubMed1995

Use of structural comparisons to select mutagenic targets in aspartate-beta-semialdehyde dehydrogenase.

L-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) from Escherichia coli has been probed by site-directed mutagenesis to identify residues that play an important function in the catalytic activity of the enzyme. Sequence homology searching among ASA DHs that have been isolated from other species and comparisons with the structures of functionally similar D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) that have been solved from several species have been utilized to select appropriate targets for mutagenesis. A highly conserved active site glutamine has been identified in the E. coli ASA DH that enhances the reactivity of the enzyme. Alteration of this residue leads to an enzyme with reduced catalytic efficiency, yet with an unchanged binding affinity for substrates and coenzyme. Replacement of an arginine residue that is conserved throughout the ASA DH and GAPDH enzyme families leads to a significant decrease in catalytic turnover and is the only mutation examined that also results in a decreased affinity for the substrates of the reaction. This residue is assigned a role in the binding of the substrate aspartate-beta-semialdehyde. Sequence alignment of ASA DH with other NADP- and NAD-dependent enzymes has resulted in the identification of a putative pyridine nucleotide binding region. Substitution of two amino acids in this region with neutral or positively charged side chains has resulted in a change in enzyme specificity. For wild-type ASA DH, NADP is strongly favored as the coenzyme, while in this mutated enzyme the selectivity has been lowered by a factor of 60, and this enzyme has comparable affinities for either pyridine nucleotide.

Amino Acid Sequence↗

PubMed2006

The Histone Database: a comprehensive resource for histones and histone fold-containing proteins.

The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.

Amino Acid Sequence↗

PubMed2001

Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase.

A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK(a1)=4.9 and pK(a2)=7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K(m) values were obtained for different p-nitrophenyl beta-glycosides and K(i) values were determined for a range of alkyl beta-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E(187) (proton donor) and E(399) (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.

Amino Acid Sequence↗

PubMed2003

The dog genome: survey sequencing and comparative analysis.

A survey of the dog genome sequence (6.22 million sequence reads; 1.5x coverage) demonstrates the power of sample sequencing for comparative analysis of mammalian genomes and the generation of species-specific resources. More than 650 million base pairs (>25%) of dog sequence align uniquely to the human genome, including fragments of putative orthologs for 18,473 of 24,567 annotated human genes. Mutation rates, conserved synteny, repeat content, and phylogeny can be compared among human, mouse, and dog. A variety of polymorphic elements are identified that will be valuable for mapping the genetic basis of diseases and traits in the dog.

Animals↗

PubMed1994

Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase.

Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.

Amino Acid Sequence↗

PubMed2004

Crassostrea gigas ferritin: cDNA sequence analysis for two heavy chain type subunits and protein purification.

Ferritin has been shown as being the principal iron storage in the majority of living organisms. In marine species, ferritin is also involved in high-level accumulation of (210)Po. As part of our work on the investigation of these radionuclides' concentration in natural environment, ferritin was searched at the gene and protein level. Ferritin was purified from the visceral mass of the oyster Crassostrea gigas by ion-exchange chromatography and HPLC. SDS-PAGE revealed one band of 20 kDa. An Expressed Sequence Tag (EST) library was screened and led to the identification of two complementary DNA (cDNA) involved in ferritin subunit expression. The complete coding sequences and the untranslated regions (UTRs) of the two genes were obtained and a 5' Rapid Amplification of cDNA Ends (RACE) was used to obtain the two iron-responsive elements (IREs) with the predicted stem-loop structures usually present in the 5'-UTR of ferritin mRNA. Sequence alignment in amino acid of the two new cDNA showed an identity with Pinctada fucata (85.4-88.3%), Lymnaea stagnalis (79.3-82.2%) and Helix pomatia (79.1-79.1%). The residues responsible for the ferroxidase center, conserved in all vertebrate H-ferritins, are present in the two oyster ferritin subunits. Oyster ferritins do not present the special characteristics of other invertebrate ferritins like insect ferritins but have some functional similarities with the vertebrate H chains ferritin.

Amino Acid Sequence↗

PubMed2001

Cloning and expression of the human single copy homologue of the mouse zinc finger protein zfr.

A human homologue of the murine zinc finger protein zfr is transcriptionally induced in the Epstein-Barr virus-positive Burkitt lymphoma cell line Raji upon treatment with the granulocyte/macrophage lineage ganglioside IV(3)NeuAc-nLcOse(4)Cer. The gene was cloned by a rapid amplification of cDNA ends approach based on a cDNA clone. The resulting hzfr sequence is 3393 base pairs in length coding for a protein of 1057 amino acids. Sequence alignments between hzfr and zfr reveal an identity of 92% on the nucleotide level and an identity of 96.4% on the amino acid level, respectively. Based on Southern blot data hzfr can be addressed as a single copy gene. Tissue-specific expression was determined by semi-quantitative PCR of normalized cDNA populations from various human tissues with glyceraldehyde-3-phosphate dehydrogenase as an internal control. Highest levels of transcripts were found in brain. hzfr transcripts could not be detected in skeletal muscle.

Amino Acid Sequence↗

PubMed2005

Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus.

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 A (1 A=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339-Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

Amino Acid Sequence↗

PubMed1998

Roles of conserved residues in the arginase family.

Arginases and related enzymes metabolize arginine or similar nitrogen-containing compounds to urea or formamide. In the present report a sequence alignment of 31 members of this family was generated. The alignment, together with the crystal structure of rat liver arginase, allowed the assignment of possible functional or structural roles to 32 conserved residues and conservative substitutions. Two of these residues were previously identified as functionally essential by analysis of inherited defects in the type I arginase gene. Nearly half of the conserved residues are either glycines or prolines located at critical bends in the protein structure. Most metal-coordinating residues, including one histidine and four aspartic acid residues, are strictly conserved. Two additional histidines involved in metal-binding and catalysis are conserved in all arginases and in almost all other family members. Two positions with invariant similarities may serve as indirect metal ligands. Evolutionary relationships within this family were also suggested. Vertebrate type I and II arginases appear to have developed independently from an early gene duplication event. A ureohydrolase sequence from Caenorhabditis elegans is more closely related to other arginases than previously appreciated, while unclassified enzymes from Methanococcus jannaschii and Methanothermus fervidus appear more similar to arginase-related enzymes. In addition, enzymes from Arabidopsis thaliana and Synechocystis, previously identified as arginases, more closely resemble arginase-related enzymes than currently known arginases.

Amino Acid Sequence↗

PubMed1999

The structure of a Staphylococcus aureus leucocidin component (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore-forming toxins.

BACKGROUND: Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS: The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS: The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer.

Amino Acid Sequence↗

PubMed2003

Enoyl-CoA hydratase. reaction, mechanism, and inhibition.

Enoyl-CoA hydratase (ECH) catalyzes the second step in the physiologically important beta-oxidation pathway of fatty acid metabolism. This enzyme facilitates the syn-addition of a water molecule across the double bond of a trans-2-enoyl-CoA thioester, resulting in the formation of a beta-hydroxyacyl-CoA thioester. The catalytic mechanism of this proficient enzyme has been studied in great depth through a combination of kinetic, spectroscopic, and structural techniques, and is proposed to occur via the formation of a single transition state. Sequence alignment and mutagenesis studies have implicated the key residues important for catalysis: Gly-141, Glu-144, and Glu-164 (rat liver ECH numbering). The two catalytic glutamic acid residues are believed to act in concert to activate a water molecule, while Gly-141 is proposed to be involved in substrate activation. Recently, two potent inhibitors of ECH have been reported in the literature, which result in the irreversible inactivation of the enzyme via covalent adduct formation. This review summarizes studies on the structure, mechanism, and inhibition of ECH.

Amino Acid Sequence↗