Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

One or more of the five acidic amino-terminal residues of skeletal muscle actin have been implicated as being important in a number of actin-related processes. We have constructed a series of actins containing mutations at Asp3 and Asp11 and tested these mutant proteins for their ability to bind to DNase I-agarose, polymerize with rabbit skeletal muscle actin, undergo amino-terminal processing, and bind to the myosin-S1 subfragment. The mutant actins were expressed in vitro using a coupled transcription/translation system which involves the synthesis of mutant RNAs with SP6 RNA polymerase followed by their translation in a rabbit reticulocyte lysate. When Asp3 was changed to Ala, His, or Asn there was no difference in the tested properties as compared to wild type actin. These results suggest that an acidic residue at position 3 is not critical for the actin functions measured. When Asp11 was changed to Glu, Asn, or His or if the conserved Asp-Asn sequence at positions 11 and 12 was reversed, the mutants were able to copolymerize with rabbit skeletal muscle actin and be cross-linked to myosin-S1 to nearly the same extent as wild type actin. However, the amount of in vitro-synthesized actin capable of binding to DNase I-agarose with high affinity or undergoing amino-terminal processing was reduced significantly relative to the wild type actin synthesized in vitro. The Asp11 mutants ran anomalously on native polyacrylamide gels suggestive of a conformational change induced in the actin. Together, these results suggest that Asp11 may be important in proper actin folding and function.
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PMID:Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis. 319 44

The three-dimensional structure of bovine pancreatic deoxyribonuclease I (DNase I) has been determined at 2.5 A resolution by X-ray diffraction from single crystals. An atomic model was fitted into the electron density using a graphics display system. DNase I is an alpha, beta-protein with two 6-stranded beta-pleated sheets packed against each other forming the core of a 'sandwich'-type structure. The two predominantly anti-parallel beta-sheets are flanked by three longer alpha-helices and extensive loop regions. The carbohydrate side chain attached to Asn 18 is protruding by approximately 15 A from the otherwise compact molecule of approximate dimensions 45 A X 40 A. The binding site of CA2+-deoxythymidine-3',5'-biphosphate (Ca-pdTp) has been determined by difference Fourier techniques confirming biochemical results that the active centre is close to His 131. Ca-pdTp binds at the surface of the enzyme between the two beta-pleated sheets and seems to interact with several charged amino acid side chains. Active site geometry and folding pattern of DNase I are quite different from staphylococcal nuclease, the only other Ca2+-dependent deoxyribonuclease whose structure is known at high resolution. The electron density map indicates that two Ca2+ ions are bound to the enzyme under crystallization conditions.
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PMID:Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution. 649 35

The autogenously regulated gene pir of Escherichia coli plasmid R6K encodes the replication protein pi. This protein binds to two sites in the operator region of the pir gene: a 22-base pair nonpalindromic sequence and a pair of palindromic 9-base pair sequences. These pi-binding sites are similar, suggesting that pi uses a single DNA-binding domain in recognizing them. We devised a plasmid system permitting isolation of mutants of the pi protein which are altered in autoregulation. A Ser87 to Asn substitution in one such mutant, designated pi 87, reduces the protein's ability to repress the pir gene promoter in vivo. DNase I protection and gel retardation assays were carried out with highly purified pi 87 protein. In these studies pi 87 exhibited altered binding to the palindromic but not to the nonpalindromic part of the operator of the pir gene. Chemical cross-linking and gel filtration analyses have shown that the dimerization properties of wild type pi and pi 87 proteins are similar in solution. We propose that the interaction of pi protein with the palindromic part of the pir operator is essential for autoregulation; we also propose that there is a fundamental difference in the mechanisms of pi protein recognition of palindromic and nonpalindromic sequences.
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PMID:Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites. 840 40

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a -10 hexamer but lacks a recognizable -35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in alpha or sigma70. As predicted if alpha were binding to Pm, we detected a polymerase-dependent footprint in the -60 region. Reconstituted RNA polymerases containing Ala substitutions in the alpha C-terminal domain were used to assay Mor-dependent transcription from Pm in vitro. The D258A substitution and alpha deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both alpha and sigma70; consistent with a key role of alpha-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) alpha-D258 is a Mor "contact site"; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via alpha-DNA contact.
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PMID:Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both alpha and sigma70 subunits of Escherichia coli RNA polymerase. 894 97

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA-cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.
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PMID:Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis. 923 Jan 29

The secretory glycoprotein DNase I acquires mannose 6-phosphate moieties on its Asn-linked oligosaccharides, indicating that it is a substrate for UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase) (Cacia, J., Quan, C., and Frenz, J. (1995) Glycobiology 4, 99). Phosphotransferase recognizes a conformation-dependent protein determinant that is present in lysosomal hydrolases, but absent in most secretory glycoproteins. To identify the amino acid residues of DNase I that are required for interaction with phosphotransferase, wild-type and mutant forms of bovine DNase I were expressed in COS-1 cells and the extent of oligosaccharide phosphorylation determined. Phosphorylation of DNase I oligosaccharides decreased from 12.6% to 2.3% when Lys-50, Lys-124, and Arg-27 were mutated to alanines, indicating that these residues are required for the basal level of phosphorylation. Mutation of lysines at other positions did not impair phosphorylation, demonstrating the selectivity of this process. When Arg-27 was replaced with a lysine, phosphorylation increased to 54%, showing that phosphotransferase prefers lysine residues to arginines. Mutation of Asn-74 to a lysine also increased phosphorylation to 50.3%, and the double mutant (R27K/N74K) was phosphorylated 79%, equivalent to the values obtained with lysosomal hydrolases. Interestingly, Lys-27 and Lys-74 caused selective phosphorylation of the neighboring Asn-linked oligosaccharide. Finally, mutation of Lys-117 to an alanine stimulated phosphorylation, demonstrating that some residues may be negative regulators of this process. We conclude that selected lysine and arginine residues on the surface of DNase I constitute the major elements of the phosphotransferase recognition domain present on this secretory glycoprotein.
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PMID:The phosphorylation of bovine DNase I Asn-linked oligosaccharides is dependent on specific lysine and arginine residues. 923 40

The bovine pancreatic (bp-) DNase I gene has been cloned from bp-cDNA and expressed in E. coli. A polynucleotide sequence of 1295 base pairs was deduced from clones of the cDNA. The sequence showed an open reading frame which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pET-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleotide coding for Met-Ala at the 5'-end. The plasmid was transformed into E. coli strain DH5alpha and the active recombinant bovine (rb-) DNase I was produced after induction of protein synthesis. From the induced culture medium, rb-DNase I was purified by chromatography on a Mono Q column. The purified rb-DNase I showed a molecular mass of 29 kDa and had the same specific activity as bp-DNase I. The NH2-terminus of rb-DNase I was Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bp-DNase I, Asn was not glycosylated.
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PMID:Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme. 946 31

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.
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PMID:Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence. 954 95

Detection of recurring three-dimensional side-chain patterns is a potential means of inferring protein function. This paper presents a new method for detecting such patterns and discusses various implications. The method allows detection of side-chain patterns without any prior knowledge of function, requiring only protein structure data and associated multiple sequence alignments. A recursive, depth-first search algorithm finds all possible groups of identical amino acids common to two protein structures independent of sequence order. The search is highly constrained by distance constraints, and by ignoring amino acids unlikely to be involved in protein function. A weighted root-mean-square deviation (RMSD) between equivalenced groups of amino acids is used as a measure of similarity. The statistical significance of any RMSD is assigned by reference to a distribution fitted to simulated data. Searches with the Ser/His/Asp catalytic triad, a His/His porphyrin binding pattern, and the zinc-finger Cys/Cys/His/His pattern are performed to test the method on known examples. An all-against-all comparison of representatives from the structural classification of proteins (SCOP) is performed, revealing several new examples of evolutionary convergence to common patterns of side-chains within different tertiary folds and in different orders along the sequence. These include a di-zinc binding Asp/Asp/His/His/Ser pattern common to alkaline phosphatase/bacterial aminopeptidase, and an Asp/Glu/His/His/Asn/Asn pattern common to the active sites of DNase I and endocellulase E1. Implications for protein evolution, function prediction and the rational design of functional regulators are discussed.
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PMID:Detection of protein three-dimensional side-chain patterns: new examples of convergent evolution. 964 96


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