Localization of the essential histidine and carboxylate group in d-xylose isomerases

D-Xylose isomerases from different bacterial strains were chemically modified with histidine and carboxylate-specific reagents. The active-site residues were identified by amino acid sequence analysis of peptides recognized by differential peptide mapping on ligand-protected and unprotected derivatized enzyme. Both types of modified residues were found to cluster in a region with consensus sequence: Phe-His-Asp-Xaa-Asp-Xaa-Xaa-Pro-Xaa-Gly, conserved in all D-xylose isomerases studied so far. These results are consistent with the recently published X-ray data of the enzyme active centre from Streptomyces rubiginosus showing hydrogen bond formation between Asp-57 and His-54 which locks the latter in one tautomeric form. A study of the pH-dependence of the kinetic parameters suggests the participation of a histidine group in the substrate-binding but not in the isomerization process. Comparison of the N-terminal amino acid sequences of several D-xylose isomerases further revealed a striking homology among the Actinomycetaceae enzymes and identifies them as a specific class of D-xylose isomerases.

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Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, Anguilla japonica

Open Life Sciences ◽

10.2478/s11535-011-0042-8 ◽

2011 ◽

Vol 6 (4) ◽

pp. 545-557 ◽

Cited By ~ 2

Author(s):

Malay Choudhury ◽

Takahiro Oku ◽

Shoji Yamada ◽

Masaharu Komatsu ◽

Keita Kudoh ◽

...

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Consensus Sequence ◽

Structural Features ◽

Lipid Binding ◽

Japanese Eel ◽

Amino Acid Sequences ◽

Novel Genes ◽

Isolation And Characterization

AbstractApolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.

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Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies

Biochemical Journal ◽

10.1042/bj2560453 ◽

1988 ◽

Vol 256 (2) ◽

pp. 453-459 ◽

Cited By ~ 21

Author(s):

O Nakanishi ◽

Y Homma ◽

H Kawasaki ◽

Y Emori ◽

K Suzuki ◽

...

Keyword(s):

Amino Acid ◽

Phospholipase C ◽

Rat Liver ◽

Peptide Mapping ◽

Polyacrylamide Gel Electrophoresis ◽

Liver Homogenate ◽

Amino Acid Sequences ◽

Dependent Manner ◽

Phosphatidylinositol 4 ◽

Hydrolysis Of

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].

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A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

Biochemical Journal ◽

10.1042/bj1290821 ◽

1972 ◽

Vol 129 (4) ◽

pp. 821-830 ◽

Cited By ~ 28

Author(s):

P. A. Gainey ◽

T. C. Pestell ◽

C. F. Phelps

Keyword(s):

Amino Acid ◽

Amino Acid Analysis ◽

Peptide Mapping ◽

Ph Dependence ◽

Glucose Dehydrogenase ◽

Group Analysis ◽

Subunit Structure ◽

Uridine Diphosphate ◽

Thiol Groups ◽

Thiol Reactivity

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ‘mapping’ in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). 5. NAD+, UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5′-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4–8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.

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Cytotoxic Necrotizing Factor Type 2 Produced by Pathogenic Escherichia coli Deamidates a Gln Residue in the Conserved G-3 Domain of the Rho Family and Preferentially Inhibits the GTPase Activity of RhoA and Rac1

Infection and Immunity ◽

10.1128/iai.67.12.6550-6557.1999 ◽

1999 ◽

Vol 67 (12) ◽

pp. 6550-6557 ◽

Cited By ~ 18

Author(s):

Motoyuki Sugai ◽

Kiyotaka Hatazaki ◽

Akira Mogami ◽

Hiroyuki Ohta ◽

Sylvie Y. Pérès ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Cultured Cells ◽

Peptide Mapping ◽

Small Gtpases ◽

Amino Acid Sequences ◽

Gtpase Activity ◽

Toxic Activity ◽

Cytotoxic Necrotizing Factor ◽

Rho Family

ABSTRACT Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122RhoGAP. Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.

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Homologues of insulinase, a new superfamily of metalloendopeptidases

Biochemical Journal ◽

10.1042/bj2750389 ◽

1991 ◽

Vol 275 (2) ◽

pp. 389-391 ◽

Cited By ~ 76

Author(s):

N D Rawlings ◽

A J Barrett

Keyword(s):

Escherichia Coli ◽

Statistical Analysis ◽

Amino Acid ◽

Consensus Sequence ◽

Amino Acid Sequences

On the basis of a statistical analysis of an alignment of the amino acid sequences, a new superfamily of metalloendopeptidases is proposed, consisting of human insulinase, Escherichia coli protease III and mitochondrial processing endopeptidases from Saccharomyces and Neurospora. These enzymes do not contain the ‘HEXXH’ consensus sequence found in all previously recognized zinc metalloendopeptidases.

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Localization and Characterization of Two Novel Genes Encoding Stereospecific Dioxygenases Catalyzing 2(2,4-Dichlorophenoxy)propionate Cleavage in Delftia acidovorans MC1

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5357-5365.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5357-5365 ◽

Cited By ~ 36

Author(s):

Kathleen M. Schleinitz ◽

Sabine Kleinsteuber ◽

Tatiana Vallaeys ◽

Wolfgang Babel

Keyword(s):

Amino Acid ◽

Ralstonia Eutropha ◽

Single Copy ◽

Amino Acid Sequences ◽

Sequence Motif ◽

Bacterial Strains ◽

Novel Genes ◽

Delftia Acidovorans ◽

Important Amino Acid ◽

Genes Encoding

ABSTRACT Two novel genes, rdpA and sdpA, encoding the enantiospecific α-ketoglutarate dependent dioxygenases catalyzing R,S-dichlorprop cleavage in Delftia acidovorans MC1 were identified. Significant similarities to other known genes were not detected, but their deduced amino acid sequences were similar to those of other α-ketoglutarate dioxygenases. RdpA showed 35% identity with TauD of Pseudomonas aeruginosa, and SdpA showed 37% identity with TfdA of Ralstonia eutropha JMP134. The functionally important amino acid sequence motif HX(D/E)X23-26(T/S)X114-183HX10-13R/K, which is highly conserved in group II α-ketoglutarate-dependent dioxygenases, was present in both dichlorprop-cleaving enzymes. Transposon mutagenesis of rdpA inactivated R-dichlorprop cleavage, indicating that it was a single-copy gene. Both rdpA and sdpA were located on the plasmid pMC1 that also carries the lower pathway genes. Sequencing of a 25.8-kb fragment showed that the dioxygenase genes were separated by a 13.6-kb region mainly comprising a Tn501-like transposon. Furthermore, two copies of a sequence similar to IS91-like elements were identified. Hybridization studies comparing the wild-type plasmid and that of the mutant unable to cleave dichlorprop showed that rdpA and sdpA were deleted, whereas the lower pathway genes were unaffected, and that deletion may be caused by genetic rearrangements of the IS91-like elements. Two other dichlorprop-degrading bacterial strains, Rhodoferax sp. strain P230 and Sphingobium herbicidovorans MH, were shown to carry rdpA genes of high similarity to rdpA from strain MC1, but sdpA was not detected. This suggested that rdpA gene products are involved in the degradation of R-dichlorprop in these strains.

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The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group

Biochemical Journal ◽

10.1042/bj1630309 ◽

1977 ◽

Vol 163 (2) ◽

pp. 309-316 ◽

Cited By ~ 2

Author(s):

I A Simpson ◽

M R Hollaway ◽

J Beard

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Amino Acid Sequence ◽

Tryptic Peptide ◽

Peptide Mapping ◽

Thiol Group ◽

Cysteine Residue ◽

Amino Acid Sequences ◽

Class I ◽

Subunit Structure

1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide ‘mapping’ it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, ‘buried’ cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.

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Correlated mutations in hydroxysteroid dehydrogenases family

Bio-Algorithms and Med-Systems ◽

10.1515/bams-2016-0024 ◽

2017 ◽

Vol 13 (1) ◽

Author(s):

Agata Żyźniewska ◽

Jacek Leluk ◽

Gabriela Żaroffe

Keyword(s):

Amino Acid ◽

Consensus Sequence ◽

Hydroxysteroid Dehydrogenase ◽

Amino Acid Sequences ◽

Multiple Sequence ◽

Hydroxysteroid Dehydrogenases ◽

Uniprot Database ◽

Correlated Mutations ◽

Almost All ◽

Degree Of Similarity

AbstractBackgroundHydroxysteroid dehydrogenase enzymes belong to the short-chain dehydrogenase/reductase (SDR) superfamily and aldo-keto reductases (AKRs). SDR is involved in the metabolism of many compounds (hormones, lipids, etc.) and is present in almost all studied genomes. Two hundred members of hydroxysteroid dehydrogenases have been analysed in terms of natural mutational variability. The second superfamily comprises AKR superfamily group enzymes whose function is catalysing the oxidation and reduction of many substrates by binding NAD(P)H as a cofactor. This kind of study is the first approach for the hydroxysteroid dehydrogenase family. This information grants practical meaning to designing potential specific drugs to fight specific diseases caused by mutations.MethodsIn the research, amino acid sequences of representatives of the hydroxysteroid dehydrogenase family were extracted from the UniProt database. In total, the analysed 200 sequences with the highest degree of similarity were shown by BLAST searches. In the sequence analyses, we used the following software: ClustalX (multiple sequence alignment), Consensus Constructor (creating consensus sequence), and CORM (finding correlated mutations).ResultsThe CORM program identified potential sites of correlated mutations in hydroxysteroid dehydrogenases. This program generated 18 tables of results that contain the amino acid positions of mutations. Seven of these are presented in this paper.ConclusionsThe primary structure of the hydroxysteroid dehydrogenase family shows high variation.

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A new member of the chalcone synthase (CHS) family in sugarcane

Genetics and Molecular Biology ◽

10.1590/s1415-47572001000100034 ◽

2001 ◽

Vol 24 (1-4) ◽

pp. 257-261 ◽

Cited By ~ 4

Author(s):

Miriam G.G. Contessotto ◽

Claudia B. Monteiro-Vitorello ◽

Pilar D.S.C. Mariani ◽

Luiz L. Coutinho

Keyword(s):

Amino Acid ◽

Active Site ◽

Chalcone Synthase ◽

Expressed Sequence Tag ◽

Consensus Sequence ◽

Stilbene Synthase ◽

Amino Acid Sequences ◽

Group 2 ◽

Expressed Sequence ◽

Group 1

Sequences from the sugarcane expressed sequence tag (SUCEST) database were analyzed based on their identities to genes encoding chalcone-synthase-like enzymes. The sorghum (Sorghum bicolor) chalcone-synthase (CHS, EC 2.3.1.74) protein sequence (gi|12229613) was used to search the SUCEST database for clusters of sequencing reads that were most similar to chalcone synthase. We found 121 reads with homology to sorghum chalcone synthase, which we were then able to sort into 14 clusters which themselves were divided into two groups (group 1 and group 2) based on the similarity of their deduced amino acid sequences. Clusters in group 1 were more similar to the sorghum enzyme than those in group 2, having the consensus sequence of the active site of chalcone and stilbene synthase. Analysis of gene expression (based on the number of reads from a specific library present in each group) indicated that most of the group 1 reads were from sugarcane flower and root libraries. Group 2 clusters were more similar to the amino acid sequence of an uncharacterized pathogen-induced protein (PI1, gi|9855801) from the S. bicolor expressed sequence tag (EST) database. The group 2 clusters sequences and PI1 proteins are 90% identical, having two amino acid changes at the chalcone and stilbene synthase consensi but conserving the cysteine residue at the active site. The PI1 EST has not been previously associated with chalcone synthase and has a different consensus sequence from the previously described chalcone synthase of sorghum. Most of the group 2 reads were from libraries prepared from sugarcane roots and plants infected with Herbaspirillum rubrisubalbicans and Gluconacetobacter diazotroficans. Our results indicate that we have identified a sugarcane chalcone synthase similar to the pathogen-induced PI1 protein found in the sorghum cDNA libraries, and it appears that both proteins represent new members of the chalcone and stilbene synthase super-family.

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Identification of calreticulin isoforms in the central nervous system

Biochemical Journal ◽

10.1042/bj2870579 ◽

1992 ◽

Vol 287 (2) ◽

pp. 579-581 ◽

Cited By ~ 18

Author(s):

S Treves ◽

F Zorzato ◽

T Pozzan

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Amino Acid ◽

Xenopus Laevis ◽

Consensus Sequence ◽

Amino Acid Level ◽

Amino Acid Sequences ◽

Open Questions ◽

Mammalian Liver ◽

The Central Nervous System

In the present paper we report the cloning and sequencing of the cDNA encoding two calreticulin isoforms from Xenopus laevis central nervous system. The two isoforms display 93% identity at the amino acid level. The predicted amino acid sequences of the amphibian calreticulins are very similar (76%) to those of mammalian liver and skeletal muscle. Xenopus laevis calreticulins are characterized by a very acidic c-terminal domain endowed with the endoplasmic-reticulum retention signal KDEL. The cDNAs of both clones encode an N-glycosylation consensus sequence. A third clone of calreticulin was also identified. The restriction map of this clone was clearly distinct from that of the two sequenced clones. These results indicate the existence of multiple calreticulin isoforms in the central nervous system and open questions about their functional role in different cells and/or subcellular compartments.

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