Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The conversion of trypsin into a protease with chymotrypsin-like activity and specificity required substitution of fifteen residues in the S1 site and two surface loops with their chymotrypsin counterparts [Hedstrom,L., Szilagyi,L. and Rutter,W.J. (1992) Science, 255, 1249-1253]. These residues may define a set of general structural determinants of specificity in the trypsin family. In order to test this hypothesis, we have attempted to convert trypsin into a protease with specificity for substrates containing small aliphatic residues by replacing the S1 site and these surface loops with the analogous residues of elastase. Five elastase-like mutant enzymes were constructed with various combinations of these substitutions. Four mutant enzymes catalyze the hydrolysis of MeOSuc-Ala-Ala-Pro-Ala-SBzl more efficiently than the hydrolysis of Suc-Ala-Ala-Pro-Phe-SBzl. This observation indicates that the mutant enzymes have elastase-like esterase specificity. The best mutant, Tr-->E1-2, is a more specific esterase than elastase: the ratio of the values of kcat/Km for MeOSuc-Ala-Ala-Pro-Ala-SBzl and Suc-Ala-Ala-Pro-Phe-SBzl is greater than 160 for Tr-->E1-2 and 50 for elastase. However, the esterase activity of Tr-->E1-2 is 300-fold less than elastase; in addition, Tr-->E1-2 has no measurable amidase activity. Thus these substitutions do not construct a protease with elastase-like activity. These experiments indicate that a unique structural solution is required for each different specificity. Previous work suggested that instability of the S1 site is a major barrier to redesigning the specificity of trypsin. This view is corroborated by preliminary structural studies of Tr-->E1-2. One dimensional 1H NMR spectrum of Tr-->E1-2 suggests that the S1 site and the two surface loops of this mutant trypsin may be disordered.
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PMID:Converting trypsin to elastase: substitution of the S1 site and adjacent loops reconstitutes esterase specificity but not amidase activity. 974 19

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ss-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ss-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Galalpha1,2Man-ol (2A) and Manalpha1, 2Man-ol (2B). The Hex2Hexitol pool contained two components, Galalpha1,2Manalpha1,2Man-ol (3A) and Manalpha1,2Manalpha1,2Man-ol (3B). The two Hex3Hexitol components were Galalpha1,2(Galalpha1, 3)Manalpha1,2Man-ol (4A) and Manalpha1,2(Galalpha1,3)Manalpha1, 2Man-ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Galalpha1,2(Galalpha1,3)Manalpha1, 2Manalpha1,2Man-ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693-2707) and gth1 (T. G. Chappell personal communication) alpha1, 2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal alpha1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the alpha1,2-linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding alpha1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alpha1,6-Man outerchain, nor the O-linked branch-forming alpha1,3-linked Gal. Furthermore, the presence of Hexalpha1,2(Galalpha1,3)Manalpha1,2- structures in the O-linked glycans implies the presence of a novel branch-forming alpha1,3-galactosyltransferase in S.pombe.
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PMID:Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides. 1020 83

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L.
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PMID:NMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains. 1265 66

Mycothiol is an abundant small molecular weight thiol found only in actinomycetes, which include mycobacteria. Mycothiol biosynthetic and detoxification enzymes are novel and unique to actinomycetes, thereby representing potential antimycobacterial targets. To better guide inhibitor design, we have determined by NMR the solution conformations of mycothiol bimane (MSmB) and the pseudodisaccharide 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins (D-GI), molecules that represent the natural substrates for the mycothiol-dependent detoxification enzyme mycothiol-S-conjugate amidase (MCA) and the mycothiol biosynthetic enzyme D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins deacetylase (AcGI deacetylase), respectively. Comparison of the mean structure of MSmB and the energy-minimized structures of two competitive spiroisoxazoline-containing MCA inhibitors shows striking similarities between these molecules in the region of the scissile amide bond of MSmB and provides structural evidence that those inhibitors are substrate mimics. Owing to our earlier finding that AcGI deacetylase will not deacetylate the unnatural isomer 1-d-GlcNAc-alpha-(1 --> 1)-L-myo-Ins (L-GI), the solution conformation of L-GI was also determined. The interglycosidic bond angles for all three compounds are comparable. When considered together with the observation that a simplified cyclohexyl thioglycoside mycothiol analogue is a good substrate for MCA, it appears that the stereochemistry of the inositol ring is critical for deacetylase function, superceding the importance of the full complement of hydroxyl groups on the "nonreducing" ring.
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PMID:Solution conformations of mycothiol bimane, 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins and 1-D-GlcNAc-alpha-(1 --> 1)-L-myo-Ins. 1271 35

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+ N-acetylmuramyl-L-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wild-type activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes.
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PMID:Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-L-alanine amidase. 1450 60

The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate at a rate of 0.9 micromol mg(-1) min(-1). The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by (13)C NMR spectroscopy. The protein contains 1 eq of Zn(2+)/subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.
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PMID:Evolution of vitamin B2 biosynthesis: structural and functional similarity between pyrimidine deaminases of eubacterial and plant origin. 1520 17

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N (4)-(N-acetyl-beta -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and (1)H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), and two xylosylated structures, Xyl(1)Man(3)GlcNAc(2) and Xyl(1)Man(4)GlcNAc(2). The PNGase A-sensitive N-glycans, containing (alpha 1-3)-linked fucose, were identified as Fuc(1)Xyl(1)Man(2)GlcNAc(2) and Fuc(1)Xyl(1)Man(3)GlcNAc(2). In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (beta 1-2)-linked xylose- and core alpha (1-3)-linked fucose-modified N-glycan chains.
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PMID:Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds. 1700 51

Enzymes from the pentose phosphate pathway (PPP) are potential drug targets for the development of new drugs against Trypanosoma brucei, the causative agent of African sleeping disease: for instance, the 6-phosphogluconate dehydrogenase is currently studied actively for such purposes. Structural and functional studies are necessary to better characterize the associated enzymes and compare them to their human homologues, in order to undertake structure-based drug design studies on such targets. In this context, the crystal structure of 6-phosphogluconolactonase (6PGL) from T. brucei, the second enzyme from PPP, was determined at 2.1 Angstroms resolution. Comparison of its sequence and structure to other related proteins in the 6PGL family with a known structure (Thermotoga maritima Tm6GPL 1PBT and Vibrio cholerae Vc6PGL (1Y89), which have not been discussed in print), or in the glucosamine-6-phosphate-deaminase family (hexameric Escherichia coli 1DEA and monomeric Bacillus subtilis 2BKV), allowed the identification of the 6PGL active site. In addition to the analysis of the crystal structure, 3D NMR interaction studies and docking experiments are reported here. Key residues involved in substrate binding or in catalysis were identified.
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PMID:Three dimensional structure and implications for the catalytic mechanism of 6-phosphogluconolactonase from Trypanosoma brucei. 1719 81

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir, and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (k(i)). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography, and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (approximately 5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process.
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PMID:Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH). 1802 88

Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 degrees C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 +/- 3 M(-1) min (-1). MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 degrees C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h (-1), indicating that the adduct is quite stable ( t1/2 = 6.5 days). At pH 7.4 and 22 degrees C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable ( t1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure.
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PMID:Binding and hydrolysis of soman by human serum albumin. 1816 44


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