Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HYDROLASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 199 records · Page 11Linked to original sources
PubMed1994

Chromosomal mapping and expression levels of a mouse soluble epoxide hydrolase gene.

The chromosomal location of a murine soluble epoxide hydrolase gene was determined using in situ mapping, restriction fragment length polymorphism (RFLP) and simple sequence length polymorphism (SSLP) analysis. In situ hybridization to mouse metaphase chromosomes using a soluble epoxide hydrolase cDNA probe showed that soluble epoxide hydrolase maps at band D of chromosome 14. An RFLP found between Mus castaneus (CAST) and Mus musculus (MEV) was used to map the soluble epoxide hydrolase gene in CAST x MEV intersubspecific testcross progeny to 14 cM from the Np-1 locus on mouse chromosome 14. SSLP markers were then used to confirm the location of soluble epoxide hydrolase at 14.0 +/- 3.7 cM distal to Np-1 and 19.2 +/- 4.3 cM proximal to D14Mit7. This region of mouse chromosome 14 is homologous with human chromosomes 8, 13 and 14. Enzyme assays and immunoblotting results suggest significant quantitative differences in expression of soluble epoxide hydrolase among three mouse strains. Northern blotting analysis showed that soluble epoxide hydrolase mRNA levels were correlated with the relative level of soluble epoxide hydrolase enzyme activity and soluble epoxide hydrolase protein in all three mouse strains.

Animals↗
PubMed1994

Leukotriene A4 hydrolase in human skin.

The biochemical properties and immunohistochemical localization of leukotriene (LT) A4 hydrolase were investigated in human skin. The activity of LTA4 hydrolase, which catalyzes the hydrolysis of LTA4 to LTB4, the most chemotactic compound known, was detected in the 100,000 x g supernatant of homogenates of human epidermis and a transformed epidermal cell line (HSC-1). No significant LTA4 hydrolase activity was detected in human whole skin or dermis. The enzymatic properties of LTA4 hydrolase isolated from human keratinocytes and peripheral leukocytes were similar. Their activities were inhibited by bestatin and captopril, and they were completely absorbed by anti-human LTA4 hydrolase antibody. By immunoblotting analysis using this antibody, LTA4 hydrolase was detected as a 70-kDa protein in human epidermis and HSC-1 and was found to be similar to the enzyme detected in peripheral mononuclear leukocytes. In human dermis, LTA4 hydrolase was barely detected by Western blotting. On the other hand, LTA4 hydrolase was demonstrated in the cytoplasm of keratinocytes in the epidermis, and in fibroblasts, infiltrating and endothelial cells in the dermis of normal human skin by immunohistochemical analysis using the immunoperoxidase method. These results suggest that LTB4 can be generated from LTA4 by LTA4 hydrolase in keratinocytes as well as fibroblasts, infiltrating and endothelial cells in the dermis of human skin.

Blotting, Western↗
PubMed1987

Comparison of the sex and subcellular distributions, catalytic and immunochemical reactivities of hepatic epoxide hydrolases in seven mammalian species.

Sex and species differences in hepatic epoxide hydrolase activities towards cis- and trans-stilbene oxide were examined in common laboratory animals, as well as in monkey and man. In general trans-stilbene oxide was found to be a good substrate for epoxide hydrolase activity in cytosolic fractions, whereas the cis isomer was selectively hydrated by the microsomal fraction (with the exception of man, where the cytosol also hydrated this isomer efficiently). The specific cytosolic epoxide hydrolase activity was highest in mouse, followed by hamster and rabbit. Epoxide hydrolase activity in the crude 'mitochondrial' fraction towards trans-stilbene oxide was also highest in mouse and low in all other species examined. Microsomal epoxide hydrolase activity was highest in monkey, followed by guinea pig, human and rabbit, which all had similar activities. Sex differences were generally small, but where significant, male animals had higher catalytic activities than females of the same species in most cases. Antibodies raised against microsomal epoxide hydrolase purified from rat liver reacted with microsomes from all species investigated, indicating structural conservation of this protein. Antibodies directed towards cytosolic epoxide hydrolase purified from mouse liver reacted only with liver cytosol from mouse and hamster and with the 'mitochondrial' fraction from mouse in immunodiffusion experiments. Immunoblotting also revealed reaction with rat liver cytosol. The cytosolic and 'mitochondrial' epoxide hydrolases in all three mouse strains and in both sexes for each strain were immunochemically identical. The anomalies in human liver epoxide hydrolase activities observed here indicate that no single common laboratory animal is a good model for man with regard to these activities.

Animals↗
PubMed1995

Cloning and characterization of the human leukotriene A4 hydrolase gene.

The human gene encoding the bifunctional aminopeptidase and epoxide hydrolase enzyme, leukotriene A4 hydrolase (LTA4 hydrolase) has been cloned from a placental lambda phage genomic library. The gene is greater than 35 kbp and contains 19 exons ranging in size over 24-312 bp. The introns range in size over 0.26-5.7 kbp. The essential zinc-binding histidine residues and glutamate residue, which delineate the zinc-binding domain required for both enzyme activities of LTA4 hydrolase, are divided between exons 10 and 11. The LTA4 hydrolase gene was localized to chromosome 12q22 utilizing fluorescence in situ hybridization. Based on the chromosome localization and genomic DNA analysis, LTA4 hydrolase was determined to be a single-copy gene. Primer-extension analysis demonstrated that the transcription initiation site of LTA4 hydrolase mRNA is 151 nucleotides upstream of the initiator ATG. Approximately 4 kbp of 5'-flanking region of the LTA4 hydrolase gene has been obtained and sequencing of 1.4 kb of this 5'-flanking region demonstrated several transcription-factor consensus sequences, including a phorbol-ester-response element (AP2) and two xenobiotic-response elements. The cloning and characterization of the human gene for LTA4 hydrolase provides a basis for further insight into transcriptional regulation of this bifunctional enzyme and its role in various inflammatory processes.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.

Amino Acid Sequence↗
PubMed1998

Two nearly identical aromatic compound hydrolase genes in a strong polychlorinated biphenyl degrader, Rhodococcus sp. strain RHA1.

The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. The etbD2 gene was located in the vicinity of bphA gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in alpha/beta hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphD genes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against the meta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphD gene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

Amino Acid Sequence↗
PubMed2006

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis.

Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.

Animals↗
PubMed2005

Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung.

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.

Aminopeptidases↗
PubMed1995

Localization of microsomal epoxide hydrolase in normal and neoplastic human kidney.

Microsomal epoxide hydrolase is a xenobiotic metabolizing enzyme that catalyzes the conversion of toxic and carcinogenic epoxides to less toxic dihydrodiols. The cellular localization and distribution of microsomal epoxide hydrolase were investigated for the first time in normal and neoplastic human kidney. Light microscopic immunohistochemical studies using an alkaline phosphatase-anti-alkaline phosphatase technique showed that in normal kidney there was a wide distribution of epoxide hydrolase immunoreactivity. The main localization of epoxide hydrolase immunoreactivity was to the proximal and distal tubule epithelial cells. Strong epoxide hydrolase immunoreactivity was also identified in epithelium of the collecting ducts. In addition, epoxide hydrolase immunoreactivity was present in vascular endothelial cells, including endothelial cells lining glomerular capillaries. Epoxide hydrolase immunoreactivity was identified in all the renal tumors, and in each tumor immunoreactivity for epoxide hydrolase was localized to tumor cells. Immunoblotting of both normal kidney and tumor microsomes confirmed the presence of a single protein band of molecular weight 49 KD corresponding to the molecular weight of human hepatic microsomal epoxide hydrolase.

Adult↗
PubMed1990

Regulation of microsomal, xenobiotic epoxide hydrolase messenger RNA in persistent hepatocyte nodules and hepatomas induced by chemical carcinogens.

We have utilized a DNA clone complementary to epoxide hydrolase mRNA as a probe to examine the level of the mRNA in persistent hepatocyte nodules and hepatomas induced by the Solt-Farber chemical carcinogenesis procedure. Epoxide hydrolase mRNA is increased 14-fold in nodules as compared to the level in normal liver. When rats with liver nodules were administered phenobarbital, an inducer of epoxide hydrolase mRNA in normal animals, a superinduction in epoxide hydrolase mRNA was observed in the nodules (22-fold) as compared to normal liver. Surprisingly, nodule induction in conjunction with phenobarbital administration also produced marked elevation in epoxide hydrolase mRNA levels in the tissue surrounding the nodules. Using HpaII and MspI to assess the degree of methylation of CCGG sites, we have found that the epoxide hydrolase gene is hypomethylated in nodules and hepatomas compared to the gene in normal liver tissue. Phenobarbital treatment alone increased epoxide hydrolase mRNA levels but did not result in hypomethylation of the epoxide hydrolase gene. These data further support the observation that hypomethylation of specific gene sequences occurs during chemical carcinogenesis and is correlated with an elevation in the steady state level of epoxide hydrolase mRNA in persistent hepatocyte nodules.

Animals↗
PubMed1987

Structure and organization of the microsomal xenobiotic epoxide hydrolase gene.

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.

Amino Acid Sequence↗
PubMed1986

Vacuolar pH is one factor that regulates hydrolase secretion.

Lysosomal hydrolases are continually secreted by Acanthamoeba as a consequence of membrane cycling between the vacuolar compartment and the cell surface. In pinocytosing amoebae acid hydrolases can be separated into two groups on the basis of their secretion kinetics. We have previously shown that in Acanthamoeba acid hydrolases are almost exclusively restricted to a single compartment, digestive vacuoles, and that pH-dependent differential binding of hydrolases to vacuolar membrane can account for the different rates of hydrolase secretion from this compartment. In this report we show that the hydrolase secretion pattern changes and that all of the hydrolases are released with the same kinetics after phagocytosis of yeast or in growth media supplemented with ammonium acetate or chloroquine, but not after phagocytosis of polystyrene beads. The changes in the pattern of hydrolase secretion correlate with changes in vacuolar pH. The vacuolar pH of pinocytosing amoebae and amoebae saturated with beads is about 4.8. This value is increased to 6.8 by accumulation of weak bases and to about 6.1 when digestive vacuoles are saturated with yeast. These results indicate that vacuolar pH modulates hydrolase transport and secretion.

Acetates↗
PubMed1983

Effects of phenobarbital, trans-stilbene oxide, and 3-methylcholanthrene on epoxide hydrolase within centrilobular, midzonal, and periportal regions of rat liver.

The effects of phenobarbital, trans-stilbene oxide, and 3-methylcholanthrene on epoxide hydrolase (EC 3.3.2.3) within centrilobular, midzonal, and periportal hepatocytes were investigated employing rabbit anti-serum produced against rat hepatic microsomal epoxide hydrolase in unlabeled antibody peroxidase-anti-peroxidase and indirect fluorescent antibody-staining techniques. In livers of control rats, midzonal and periportal hepatocytes bound the anti-epoxide hydrolase to similar extents while centrilobular hepatocytes bound approximately 25% more antibody. 3-Methylcholanthrene did not cause significant alterations in immunohistochemical staining for epoxide hydrolase within any region of the liver lobule, whereas phenobarbital and trans-stilbene oxide produced significant alterations in both the intensity and pattern of intralobular staining for the enzyme. After 4 days of phenobarbital pretreatment, anti-epoxide hydrolase binding to hepatocytes was slightly, but significantly, elevated, especially within midzonal regions. After 7 days of phenobarbital pretreatment, anti-epoxide hydrolase binding was increased by approximately 65% within midzonal regions and by approximately 41 and 24%, respectively, within centrilobular and periportal regions. In livers of trans-stilbene oxide-pretreated rats, anti-epoxide hydrolase binding was increased by approximately 80% within both the midzonal and periportal regions and by approximately 43% within centrilobular regions. These immunohistochemical findings demonstrate that phenobarbital and trans-stilbene oxide both induce epoxide hydrolase nonuniformly within the liver lobule. However, while phenobarbital induces the enzyme to the greatest extent within midzonal hepatocytes and to the least extent within periportal hepatocytes, trans-stilbene oxide induces epoxide hydrolase equally within midzonal and periportal hepatocytes.

Animals↗
PubMed1994

Reengineering the specificity of a serine active-site enzyme. Two active-site mutations convert a hydrolase to a transferase.

Two residues are known to play important catalytic roles in fatty acyl-thioester hydrolase, thioesterase II: Ser-101, the site of a covalent acyl-enzyme intermediate, and His-237 which is within hydrogen bonding distance of Ser-101 and facilitates catalysis by increasing the nucleophilicity of this residue. In this study we have examined the effect of mutations at these two residues on the ability of the enzyme to function as a hydrolase and, in the presence of a thiol acceptor, as an acyltransferase. In the hydrolase reaction kcat values for the wild-type, H237R, S101C, and S101C, H237R thioesterase enzymes were 0.11, < 0.002, 0.10, and < 0.002 s-1, respectively, and at steady state, the proportion of each enzyme present as the covalent acyl-enzyme intermediate was 11, 91, 71, and 100%, respectively. In the acyltransferase reaction no activity could be detected for the wild-type or H237R enzymes but the specific activities of the S101C and S101C/H237R thioesterases were 170 and 1300 nmol/min/mg of protein, respectively. From this data we conclude the following: the wild-type enzyme functions exclusively as a hydrolase. The H237R mutant acts ineffectively as a hydrolase primarily because the deacylation reaction is drastically retarded. The S101C enzyme functions well as a hydrolase, even though the rate of deacylation is adversely affected, and this enzyme can also perform as an acyltransferase. Mutation of both catalytic residues leads to a complete loss of hydrolase activity and the S101C,H237R mutant functions as an effective acyltransferase exhibiting kcat values higher then those of the wild-type enzyme acting as a hydrolase. This study reveals that, with only two amino acid replacements, an enzyme capable of functioning exclusively as a hydrolase can be converted into an equally active enzyme performing solely as an acyltransferase.

Acylation↗
PubMed1992

Molecular and immunological characterization of ADP-ribosylarginine hydrolases.

Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.

Amino Acid Sequence↗
PubMed2002

Cloning and expression of the gene for soybean hydroxyisourate hydrolase. Localization and implications for function and mechanism.

The gene encoding hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme, has been cloned from soybean (Glycine max). The gene encodes a protein that is 560 amino acids in length and contains a 31-amino acid signal sequence at the N terminus that is not present in the mature protein. The presence of two SKL motifs near the C terminus suggests that the protein resides in the peroxisome. This expectation is borne out by results from immunogold electron microscopy, which revealed that hydroxyisourate hydrolase was localized in the peroxisomes of uninfected root nodules. The gene encoding hydroxyisourate hydrolase was expressed in Escherichia coli, and soluble, catalytically active enzyme was purified to homogeneity. Sequence analysis revealed considerable homology with members of the beta-glucosidase family of enzymes. Two glutamate residues, E199 and E408, align with the conserved glutamates that play catalytic roles in the beta-glucosidases. However, the other residues that have been identified by crystallography to interact directly with the substrates in beta-glucosidases are not conserved in hydroxyisourate hydrolase. The E199A and E408A hydroxyisourate hydrolase mutants were devoid of detectable catalytic activity. Analysis of transcripts for hydroxyisourate hydrolase demonstrated that its level of expression was highest in the nodule; mRNA was detectable 12 d after infection and increased until 21 d postinfection, then declined. In a similar manner, immunodetection of hydroxyisourate hydrolase indicated preferential localization in the nodule; the amount of protein detected was maximal at 21 d postinfection. The pattern of expression of hydroxyisourate hydrolase matched that of urate oxidase, and supports the hypothesis that hydroxyisourate hydrolase plays a role in ureide metabolism.

Amidohydrolases↗
PubMed1995

Detection of arginine-ADP-ribosylated protein using recombinant ADP-ribosylarginine hydrolase.

We made use of ADP-ribosylarginine hydrolase to detect arginine-ADP- ribosylated proteins. The hydrolase was expressed in Escherichia coli as a protein fused with glutathione S-transferase (GST). The fusion protein GST-ADP-ribosylarginine hydrolase catalyzed the hydrolysis of alpha-ADP-ribosylarginine to produce ADP-ribose and arginine. Casein ADP-ribosylated with [32P]NAD and chicken heterophil arginine-specific ADP-ribosyltransferase served as a substrate for the recombinant ADP-ribosylarginine hydrolase and the released ADP-ribose was determined. Protein ADP-ribosylated by cholera toxin could serve as substrate of the hydrolase but protein ADP-ribosylated by pertussis toxin, diphtheria toxin, or C(3) enzyme of Clostridium botulinum could not. The hydrolase did not release the radioactivity incorporated into isolated rat liver nuclei incubated with [(32)P]NAD or in bovine brain cytosol incubated with [(32)P]ADP-ribose. In homogenate of mouse heart which contained arginine-specific ADP-ribosyltransferase, labeling of a 55-kDa protein by incubation with [(32)P]NAD was removed by ADP-ribosylarginine hydrolase treatment; hence, the specific hydrolysis of ADP-ribose-arginine bond by GST-ADP-ribosylarginine hydrolase can be used to detect the arginine-ADP-ribosylated proteins in crude preparations. Arginine--ADP-ribosylated proteins in crude preparations. Arginine-ADP-ribosylated proteins in mouse spleen lymphocytes were identified using this method.

Adenosine Diphosphate Ribose↗
PubMed2003

Crystal structure of Escherichia coli thioesterase I/protease I/lysophospholipase L1: consensus sequence blocks constitute the catalytic center of SGNH-hydrolases through a conserved hydrogen bond network.

Escherichia coli thioesterase I (TAP) is a multifunctional enzyme possessing activities of thioesterase, esterase, arylesterase, protease, and lysophospholipase. In particular, TAP has stereoselectivity for amino acid derivative substrates, hence it is useful for the kinetic resolution of racemic mixtures of industrial chemicals. In the present work, the crystal structure of native TAP was determined at 1.9A, revealing a minimal SGNH-hydrolase fold. The structure of TAP in complex with a diethyl phosphono moiety (DEP) identified its catalytic triad, Ser10-Asp154-His157, and oxyanion hole, Ser10-Gly44-Asn73. The oxyanion hole of TAP consists of three residues each separated from the other by more than 3.5A, implying that all of them are highly polarized when substrate bound. The catalytic (His)C(epsilon1)-H...O=C hydrogen bond usually plays a role in the catalytic mechanisms of most serine hydrolases, however, there were none present in SGNH-hydrolases. We propose that the existence of the highly polarized tri-residue-constituted oxyanion hole compensates for the lack of a (His)C(epsilon1)-H...O=C hydrogen bond. This suggests that members of the SGNH-hydrolase family may employ a unique catalytic mechanism. In addition, most SGNH-hydrolases have low sequence identities and presently there is no clear criterion to define consensus sequence blocks. Through comparison of TAP and the three SGNH-hydrolase structures currently known, we have identified a unique hydrogen bond network which stabilizes the catalytic center: a newly discovered structural feature of SGNH-hydrolases. We have defined these consensus sequence blocks providing a basis for the sub-classification of SGNH-hydrolases.

Amino Acid Sequence↗