SEARCH · PubMed Health

Results for “Sequence Alignment”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,711 records · Page 95Linked to original sources

PubMed2003

GmPAP3, a novel purple acid phosphatase-like gene in soybean induced by NaCl stress but not phosphorus deficiency.

Purple acid phosphatases (PAPs) are commonly found in plants, but the physiological functions of different classes of PAPs are not thoroughly understood. In the present study, we identified a novel gene, GmPAP3, from salt-stressed soybean using suppression subtractive hybridization (SSH) techniques. Protein sequence alignment studies and phylogenetic analysis strongly suggested that GmPAP3 belongs to the group of plant PAPs and PAP-like proteins that are distinct from those of fungi and animals. In addition, the invariable consensus metal binding residues of PAPs were all conserved in GmPAP3. Surprisingly, analysis of protein sorting signals showed that a putative mitochondrion targeting transit peptide is present on GmPAP3. Northern blot analysis revealed that NaCl stress causes a general induction of GmPAP3 expression in both roots and leaves of various cultivated (Glycine max) and wild (Glycine soja) soybean varieties. Further test using two genetically unrelated cultivated soybean varieties showed that the expression pattern of GmPAP3 is distinct from other PAP genes in soybeans. NaCl stress and oxidative stress but not phosphorus (P) starvation induces the expression of GmPAP3. These results suggest that the physiological role of GmPAP3 might be related to the adaptation of soybean to NaCl stress, possibly through its involvement in reactive oxygen species (ROS) forming and/or scavenging or stress-responding signal transduction pathways.

Acid Phosphatase↗

PubMed2004

Functional characterization of a Drosophila mitochondrial uncoupling protein.

Sequence alignment of conserved signature motifs predicts the existence of the uncoupling protein 5 (UCP5)/brain mitochondrial carrier protein (BMCP1) homologue in Drosophila melanogaster. Here we demonstrate the functional characterization of the Drosophila melanogaster UCP5 protein (DmUCP5) in the heterologous yeast system, the first insect UCP reported to date. We show that physiological levels of DmUCP5 expression are responsible for an increase in state 4 respiration rates and a decrease in mitochondrial membrane potential. Furthermore, similar to UCP1, UCP2, and UCP3, the uncoupling activity of DmUCP5 is augmented by fatty acids and inhibited by the purine nucleotide GDP. Thus, DmUCP5 shares the mechanisms known to regulate the UCPs characterized to date. A lack of growth inhibition observed in DmUCP5 expressing yeast is consistent with the notion that physiological uncoupling has a minimal effect on cell growth. Finally, semiquantitative RT-PCR analysis shows a distinctive pattern of DmUCP5 expression predominantly localized in the adult head, similar to the expression pattern of its mammalian homologues. The conserved regulation of the expression of this gene from mammals to fruit flies suggests a role for UCP5 in the brain.

Amino Acid Sequence↗

PubMed1997

Three-dimensional model of sensory rhodopsin I reveals important restraints between the protein and the chromophore.

A structural model is constructed for the integral membrane protein, sensory rhodopsin I (SRI), the phototaxis receptor of the archaeon Halobacterium salinarium. The model is built on the template of the homologous bacteriorhodopsin (BR). The modeling procedure includes sequence alignment, a side chain rotamer search and simulated annealing by restricted molecular dynamics. The structure is in general agreement with previous results from mutagenesis experiments, chromophore substitution and room and cryogenic temperature spectroscopy. In particular, a residue near the beta-ionone ring of the retinylidene chromophore is found to be critical in maintaining the proper isomeric conformation of the chromophore; a layer of residues lying on the cytoplasmic side of the chromophore pocket is found to modulate the restraints around the C13 region of the chromophore, affecting the isomerizations around its 13 = 14 bond that are important to the protein's activity. The restraints in these regions are more stringent in SRI than in BR. The tightened restraints are chiefly due to van der Waals interactions, where the attractive and repulsive components play separable roles. Aromatic residues account for a majority of the restrictive interactions. It is hypothesized that the enhanced barriers due to these restrictions regulate the progress of SRI's photocycle, so that it can couple with the phototaxis reaction chain in the bacterium. A possibility is also suggested that conformational changes of the protein provide the signal recognized by the transducer.

Amino Acid Sequence↗

PubMed2004

Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli.

The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60 degrees C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

Aldose-Ketose Isomerases↗

PubMed2003

Substrate (aglycone) specificity of human cytosolic beta-glucosidase.

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays beta-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (beta/alpha)(8)-barrel fold characteristic of family 1 beta-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val(168)-->Tyr, Phe(225)-->Ser, Tyr(308)-->Ala and Tyr(308)-->Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val(168)-->Tyr mutation did not affect K (m) on p -nitrophenyl ( p NP)-glycosides, but increased K (m) 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe(225)-->Ser and Tyr(308)-->Ala mutations, and, to a lesser degree, the Tyr(308)-->Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.

Amino Acid Sequence↗

PubMed2003

CASTOR: clustering algorithm for sequence taxonomical organization and relationships.

Given a set of related proteins, two important problems in biology are the inference of protein subsets such that members of one subset share a common function and the identification of protein regions that possess functional significance. The former is typically approached by hierarchical bottom-up clustering based on pairwise sequence similarity and various linkage rules. The latter is typically approached in a supervised manner, based on global multiple sequence alignment. However, the two problems are inextricably linked, since functional subsets are usually characterized by distinctive functional regions. This paper introduces CASTOR, an automatic and unsupervised system that addresses both problems simultaneously and efficiently. It identifies protein regions that are likely to have functional significance by discovering and refining statistically significant motifs. It infers likely functional protein subsets and their relationships based on the presence of the discovered motifs in a top-down and recursive manner, allowing the identification of both hierarchical and nonhierarchical subset relationships. This is, to our knowledge, the first system that approaches both problems simultaneously in a top-down, systematic manner. CASTOR's performance is evaluated against the G-protein coupled receptor superfamily. The identified protein regions lead to a taxonomical organization of this superfamily that is in remarkable agreement with a biologically motivated one and which outperforms those produced by bottom-up clustering methods. We also find that conventional hierarchical representations may fail to accurately describe the complexity of evolutionary development responsible for the final organization of a complex protein family. In particular, many functional relationships governing distant subfamilies of such a protein family may not be represented hierarchically.

Algorithms↗

PubMed2002

The consensus concept for thermostability engineering of proteins: further proof of concept.

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.

6-Phytase↗

PubMed2003

[Effect of N-terminal deletion on biological activity of vascular endothelial cell growth inhibitor].

Vascular endothelial cell growth inhibitor (VEGI) is a novel cytokine which belongs to the TNF superfamily. It can inhibit the proliferation of endothelial cells and neovascularization. However, little is known about the structure-function relationship of VEGI. In order to study the effect of the N-terminus of VEGI on biological properties, the sequence alignment among VEGI and TNF superfamily members based on structure knowledge was done, and then two truncated forms of VEGI were constructed, in which 43 and 51 amino acids from N-terminus were deleted and named VEGI(131) and VEGI(123). Recombinant proteins were generated from E. coli. The expression rates were 25.2% (VEGI(131)) and 27.8% (VEGI(123)) of total bacterial proteins. After purification the purity reached 92.5% (VEGI(131)) and 91.6% (VEGI(123)). VEGI(131) showed significant inhibitory effect on growth of human umbilical vein endothelial cells (HUVEC), IC(50) of VEGI(131) being 35 mg/L. Under the same conditions, IC(50) of VEGI(151) (the wild type of VEGI) was 27 mg/L, but VEGI(123) showed no inhibitory effect. On chick choriallantic membrane (CAM) assay, VEGI(151) markedly reduced the number of main vessels, and VEGI131 decreased capillary number, while the effect of VEGI(123) was almost the same as control. These results suggest that the first 43 amino acids from N-terminus of VEGI have no significant effect on biological activity, but the amino acids 44-51 at N-terminus are required for full biological activity.

Allantois↗

PubMed2003

Residues in internal repeats of the rice cation/H+ exchanger are involved in the transport and selection of cations.

In plants, the cation/H+ exchanger (CAX) translocates Ca2+ and other metal ions into vacuoles using the H+ gradient formed by H+-ATPase and H+-pyrophosphatase. Such exchangers carrying 11 transmembrane domains (TMs) have been isolated from plants, yeast, and bacteria. In this study, multiple sequence alignment of several CAXs revealed the presence of highly conserved 36-residue regions between TM3 and TM4 and between TM8 and TM9. These two repetitive motifs are designated repeats c-1 and c-2. Using site-directed mutagenesis, we generated 31 mutations in the repeats of the Oryza sativa CAX, which translocates Ca2+ and Mn2+. Mutant exchangers were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and Mn2+ because of the absence of vacuolar Ca2+-ATPase and the Ca2+/H+ exchanger. Mutant exchangers were classified into six classes according to their tolerance for Ca2+ and Mn2+. For example, the class III mutants had no tolerance for either ion, and the class IV mutants had tolerance only for Ca2+. The biochemical function of each residue was estimated. We investigated the membrane topology of the repeats using a method combining cysteine mutagenesis and sulfhydryl reagents. Our results suggest that repeat c-1 re-enters the membrane from the vacuolar luminal side and forms a solution-accessible region. Furthermore, several residues in repeats c-1 and c-2 were found to be conserved in animal Na+/Ca2+ exchangers. Finally, we suggest that these re-entrant repeats may form a vestibule or filter for cation selection.

Amino Acid Sequence↗

PubMed2004

Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss).

A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41-45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1beta in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1beta in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1beta in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout.

Alternative Splicing↗

PubMed2005

Interactions of the HIV-1 Tat and RAP74 proteins with the RNA polymerase II CTD phosphatase FCP1.

FCP1, a phosphatase specific for the carboxyl-terminal domain of the largest subunit of RNA polymerase II, is regulated by the HIV-1 Tat protein, CK2, TFIIB, and the large subunit of TFIIF (RAP74). We have characterized the interactions of Tat and RAP74 with the BRCT-containing central domain of FCP1 (FCP1(562)(-)(738)). We demonstrated that FCP1 is required for Tat-mediated transactivation in vitro and that amino acids 562-685 of FCP1 are necessary for Tat interaction in yeast two-hybrid studies. From sequence alignments, we identified a conserved acidic/hydrophobic region in FCP1 adjacent to its highly conserved BRCT domain. In vitro binding studies with purified proteins indicate that HIV-1 Tat interacts with both the acidic/hydrophobic region and the BRCT domain of FCP1, whereas RAP74(436)(-)(517) interacts solely with a portion of the acidic/hydrophobic region containing a conserved LXXLL-like motif. HIV-1 Tat inhibits the binding of RAP74(436)(-)(517) to FCP1. In a companion paper (K. Abbott et al. (2005) Enhanced Binding of RNAPII CTD Phosphatase FCP1 to RAP74 Following CK2 Phosphorylation, Biochemistry 44, 2732-2745, we identified a novel CK2 site adjacent to this conserved LXXLL-like motif. Phosphorylation of FCP1(562)(-)(619) by CK2 at this site increases binding to RAP74(436)(-)(517), but this phosphorylation is inhibited by Tat. Our results provide insights into the mechanisms by which Tat inhibits the FCP1 CTD phosphatase activity and by which FCP1 mediates transcriptional activation by Tat. In addition to increasing our understanding of the role of HIV-1 Tat in transcriptional regulation, this study defines a clear role for regions adjacent to the BRCT domain in promoting important protein-protein interactions.

Amino Acid Sequence↗

PubMed2001

Characterization of glutathione amide reductase from Chromatium gracile. Identification of a novel thiol peroxidase (Prx/Grx) fueled by glutathione amide redox cycling.

Among the Chromatiaceae, the glutathione derivative gamma-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 microm for glutathione amide disulfide and NADH, respectively (in contrast to K(m) values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.

Amino Acid Sequence↗

PubMed2003

[Comparison of specific genomic DNA fragment between Microtus fortis calamorum and Microtus fortis fortis].

Microtus fortis(Taxonomy ID: 100897), also named as reed vole, is classified as Microtus, Micotinae, Cricetidae, Rodentia, Mammalia on taxonomy. Microtus fortis mainly distributes in China. Some areas of Russia, North Korea and Mongolia close to Northeast borderland of China also have a small number of Microtus fortis in distribution. Microtus fortis in China has principally 4 subspecies, and most of them live is the drainage area of Yangtse River. Schistosoma japonicum (one of commonly parasites in China) can infect about 40 kinds of mammalian animals, including the human being, but could not infect Microtus foris. It is known as the only animal in Dongting Lake region of China which has the ability of natural resistance to Schistosoma japonicum. The Microtus fortis domesticated in laboratory has the same biological characteristics as the wild one and these characteristics could be inherited to its progeny steadily. We got a specific DNA fragment from genomic library of Microtus fortis. This DNA fragment in genomic DNA of human beings, Kunming mice, Balb/c mice and C57BL/6J mice could not be detected by dot blot hybridization and PCR, apart from genomic DNA of Microtus fortis. In this report, the differences of genomic DNA in 34 Microtus fortis were compared between Microtus fortis calamorum(Dongting Lake region of southern China) and Microtus fortis fortis (Ningxia province of northern China). The residing localion of these two subspecies is far away about 1,200 kilometers from each other. The genomic DNA of Microtus fortis calamorum and Microtus fortis fortis were extracted and amplified by PCR according to the specific genomic DNAs sequence of Microtus fortis reported previously (Accession number in GenBank: AF277394). The amplified DNA fragments were inserted into pGEM-T easy vector and sequenced. The DNA fragment sequencing results from the two subspecies were compared to detect whether there was any difference. 19 alleles were found from Microtus fortis (20 of Microtus fortis calamorum and 14 of Microtus fortis fortis). The results of multiple sequence alignment showed that 25 single nucleotide polymorphism (SNP) sites were existed in the different Microtus fortis individuals, including transition (G-->A,A-->G,T-->C,C-->T), transversion(G-->T,A-->T,T-->A,C-->A), insertion (CA) and deletion (TGTTTT). The difference of genomic DNA from two subspecies were obvious, especially on the sites of 146, 192, 223, 224 and 235. The insertion of CA on the sites of 223 and 224 as well as A-->G transition on the site of 235 was only occurred in Microtus fortis fortis. They are not found in Microtus fortis calamorum so far. They could be divided into two groups according to phylogenetic tree analysis results, one was the genomic DNA of Microtus fortis calamorum and the other one was that of Microtus fortis fortis. However, the homologues reached up to 98% between two subspecies. These results are very important for us to further understand the genetic background, biological characteristics, evolutionary rule and the anti-schistosoma japonicum mechanism of Microtus fortis at the molecular levels. The specific base changes of the DNA fragment between the two subspecies are probably correlative with the animal immigration, survival conditions, and species evolution.

Animals↗

PubMed2003

Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol delta8-delta7 isomerase activity.

EBP (emopamil-binding protein) is a high-affinity binding protein for [3H]emopamil and belongs to the family of so-called sigma receptors. Mutations that disrupt EBP's 3beta-hydroxysteroid sterol delta8-delta7 isomerase activity (EC 5.3.3.5) impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctata. We identified a human cDNA for a novel EBPL (EBP-like protein) with a calculated mass of 23.2 kDa. Amino acid sequence alignments and phylogenetic analysis revealed that EBPL is distantly related to EBP (31% identity and 52% similarity) and found in animals but not in plants. EBPL is encoded by four exons on human chromosome 13q14.2 covering 30.7 kb, and a partially processed EBPL pseudogene was found on 16q21. The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney. Upon heterologous expression in yeast EBPL had no detectable 3beta-hydroxysteroid sterol delta8-delta7 isomerase and sigma-ligand-binding activity. Nine out of ten amino acid residues essential for catalytic activity of EBP were conserved in EBPL. Replacement of the only differing residue (EBP-Y111W) reduced catalytic activity of EBP. Transfer of the divergent residue from EBP to EBPL (EBPL-W91Y) and chimaerization of EBP and EBPL at various positions failed to restore catalytic activity of EBPL. Chemical cross-linking induced homodimerization of EBPL and EBP. Whereas mevinolin increased the mRNA for EBP and DHCR7 (delta7-sterol reductase) in HepG2 cells, it had no effect on mRNAs for EBPL and sigma1 receptor, indicating that EBP and EBPL expression are not co-ordinated. We propose that EBPL has a yet-to-be-discovered function other than cholesterol biosynthesis.

Amino Acid Sequence↗

PubMed1999

Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates.

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.

Acid Anhydride Hydrolases↗

PubMed2005

A novel pathogenesis-related protein (SsPR10) from Solanum surattense with ribonucleolytic and antimicrobial activity is stress- and pathogen-inducible.

A cDNA clone (designated as SsPR10, GenBank Accession Number AY660753 ) encoding a PR10 protein from yellow-fruit nightshade (Solanum surattense) was isolated and characterized. SsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17.58 kDa and pI of 5.29. Sequence alignments showed that SsPR10 had high identity (68.1%) with CaPR10, but had only about 31.7% identity with JIOsPR10 at the amino acid level. Genomic DNA gel blot analysis indicated that SsPR10 belonged to a multigene family. The constitutively expressed SsPR10 was detected to be the highest in roots of the sterile seedlings cultured in jars, while SsPR10 expression was the highest in old yellow leaves from the seedlings incubated with sap containing TMV. SsPR10 always expressed at slightly higher level in senescent leaves than in tender ones under both conditions. Further expression analysis revealed that the signaling components of defense/stress pathways (MeJA, SA, ABA, GA3, H2O2 and Cu2+) up-regulated significantly the SsPR10 mRNA levels over the control. However, darkness failed to induce SsPR10 expression and its expression was also inhibited by cold treatment. The SsPR10 was successfully expressed in Eschericha coli and the expressed protein was purified to near homogeneity. The dialytically renatured SsPR10 protein without phosphorylation exhibited ribonucleolytic activity against S. surattense leaf total RNA preparations and could inhibit hyphal growth of Pyricularia oryzae. Our findings suggest that the novel stress- and pathogen-inducible SsPR10 with ribonucleolytic and antimicrobial activity participates not only in the defense/stress response pathways but also in plants' growth, development and senescence.

Amino Acid Sequence↗

PubMed2003

Effects of mutagenesis of Gln97 in the switch II region of Escherichia coli elongation factor Tu on its interaction with guanine nucleotides, elongation factor Ts, and aminoacyl-tRNA.

Elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA to the A-site of the ribosome. In a multiple-sequence alignment of prokaryotic EF-Tu's, Gln97 is nearly 100% conserved. In contrast, in mammalian mitochondrial EF-Tu's, the corresponding position is occupied by a conserved proline residue. Gln97 is located in the switch II region in the GDP/GTP binding domain of EF-Tu. This domain undergoes a significant structural rearrangement upon GDP/GTP exchange. To investigate the role of Gln97 in bacterial EF-Tu, the E. coli EF-Tu variant Q97P was prepared. The Q97P variant displayed no activity in the incorporation of [(14)C]Phe on poly(U)-programmed E. coli ribosomes. The Q97P variant bound GDP more tightly than the wild-type EF-Tu with K(d) values of 7.5 and 12 nM, respectively. The intrinsic rate of GDP exchange was 2-3-fold lower for the Q97P variant than for wild-type EF-Tu in the absence of elongation factor Ts (EF-Ts). Addition of EF-Ts equalized the GDP exchange rate between the variant and wild-type EF-Tu. The variant bound GTP at 3-fold lower levels than the wild-type EF-Tu. Strikingly, the Q97P variant was completely inactive in ternary complex formation, accounting for its inability to function in polymerization. The structural basis of these observations is discussed.

Amino Acid Sequence↗

PubMed1995

Identification of the acid/base catalyst in Agrobacterium faecalis beta-glucosidase by kinetic analysis of mutants.

The catalytic mechanism of the retaining beta-glucosidase (Abg) from Agrobacterium faecalis involves a double-displacement process in which an alpha-glucosyl-enzyme intermediate is formed with general acid catalytic assistance and then hydrolyzed with general base assistance. Glu170 was identified as an important residue, possibly the acid/base catalyst, on the basis of sequence alignments. This glutamate is conserved in almost all enzymes in family 1 of glycoside hydrolases. Detailed pre-steady-state and steady-state kinetic analyses of the mutant E170G suggested very strongly that Glu170 is the acid/base catalyst. First, kcat values were invariant with pH over the range of 5.0-9.0. Secondly, rates of formation of the glycosyl-enzyme, calculated from kcat/Km and k2, were similar to those of wild-type enzyme for substrates not requiring protonic assistance but dramatically reduced for those needing acid catalysis. Thirdly, addition of azide as a competitive nucleophile increased kcat values 100-300-fold for substrates whose rate-limiting step is deglycosylation, yielding beta-glucosyl azide, but had no effect on the wild-type enzyme. Other anionic nucleophiles had similar, but less dramatic effects. Previous results [Gebler, J.C., et al. (1995) 34, 14547-14553] had indicated that Tyr298F is important for catalysis. The kinetic consequences of the mutations in the double mutant E170G-Y298F are additive, resulting in a 10(6)-fold reduction in kcat values and allowing the accumulation of a stable (t1/2 > 7 h) glucosyl-enzyme intermediate. Thus, Glu170 and Tyr298 function independently, and a possible role for Tyr298 in modulating the pKa of the catalytic nucleophile is proposed.

Amino Acid Sequence↗