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)

A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.
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PMID:Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family. 1519 15

Enterococcus faecalis lipoamidase was discovered almost 50 years ago (Reed, L. J., Koike, M., Levitch, M. E., and Leach, F. R. (1958) J. Biol. Chem. 232, 143-158) as an enzyme activity that cleaved lipoic acid from small lipoylated molecules and from pyruvate dehydrogenase thereby inactivating the enzyme. Although the partially purified enzyme was a key reagent in proving the crucial role of protein-bound lipoic acid in the reaction mechanism of the 2-oxoacid dehydrogenases, the identity of the lipoamidase protein and the encoding gene remained unknown. We report isolation of the lipoamidase gene by screening an expression library made in an unusual cosmid vector in which the copy number of the vector is readily varied from 1-2 to 40-80 in an appropriate Escherichia coli host. Although designed for manipulation of large genome segments, the vector was also ideally suited to isolation of the gene encoding the extremely toxic lipoamidase. The gene encoding lipoamidase was isolated by screening for expression in E. coli and proved to encode an unexpectedly large protein (80 kDa) that contained the sequence signature of the Ser-Ser-Lys triad amidohydrolase family. The hexa-histidine-tagged protein was expressed in E. coli and purified to near-homogeneity. The purified enzyme was found to cleave both small molecule lipoylated and biotinylated substrates as well as lipoic acid from two 2-oxoacid dehydrogenases and an isolated lipoylated lipoyl domain derived from the pyruvate dehydrogenase E2 subunit. Lipoamidase-mediated inactivation of the 2-oxoacid dehydrogenases was observed both in vivo and in vitro. Mutagenesis studies showed that the residues of the Ser-Ser-Lys triad were required for activity on both small molecule and protein substrates and confirmed that lipoamidase is a member of the Ser-Ser-Lys triad amidohydrolase family.
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PMID:Expression cloning and demonstration of Enterococcus faecalis lipoamidase (pyruvate dehydrogenase inactivase) as a Ser-Ser-Lys triad amidohydrolase. 1552 86

In Crithidia fasciculata the biosynthesis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBanktrade mark accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg(2+)-ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K(m) values for GSH, Mg(2+)-ATP, and glutathionylspermidine were 407, 222, and 480 microm, respectively, and the k(cat) was 8.7 s(-1) for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.
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PMID:Trypanothione synthesis in crithidia revisited. 1553 51

Peptide amidase (Pam), a hydrolytic enzyme that belongs to the amidase signature (AS) family, selectively catalyzes the hydrolysis of the C-terminal amide bond (CO-NH(2)) of peptides. The recent availability of the X-ray structures of Pam, fatty acid amide hydrolase, and malonamidase E2 has led to the proposal of a novel Ser-Ser-Lys catalytic triad mechanism for the amide hydrolysis by the AS enzymes. The molecular dynamics (MD) simulations using the CHARMM force field were performed to explore the catalytic mechanism of Pam. The 1.8 A X-ray crystal structure of Pam in complex with the amide analogue of chymostatin was chosen for the initial coordinates for the MD simulations. The five systems that were investigated are as follows: (i) enzyme.substrate with Lys123-NH(2), (ii) enzyme.substrate with Lys123-NH(3)(+), (iii) enzyme.substrate with Lys123-NH(3)(+) and Ser226-O(-), (iv) enzyme.transition state, and (v) enzyme.tetrahedral intermediate. Our data support the presence of the hydrogen bonding network among the catalytic triad residues, Ser226, Ser202, and Lys123, where Ser226 acts as the nucleophile and Ser202 bridges Ser226 and Lys123. The MD simulation supports the catalytic role of the crystallographic waters, Wat1 and Wat2. In all the systems that have been studied, the backbone amide nitrogens of Asp224 and Thr223 create an oxyanion hole by hydrogen bonding to the terminal amide oxygen of the substrate, and stabilize the oxyanion tetrahedral intermediate. The results from both our computational investigation and previously published experimental pH profile support two mechanisms. In a mechanism that is relevant at lower pH, the Lys123-NH(3)(+)-Ser202 dyad provides structural support to the catalytic residue Ser226, which in turn carries out a nucleophilic attack at the substrate amide carbonyl in concert with Wat1-mediated deprotonation and stabilization of the tetrahedral transition state by the oxyanion hole. In the mechanism operating at higher pH, the Lys123-NH(2)-Ser202 catalytic dyad acts as a general base to assist addition of Ser226 to the substrate amide carbonyl. The results from the MD simulation of the tetrahedral intermediate state show that both Ser202 and Lys123 are possible candidates for protonation of the leaving group, NH(2), to form the acyl-enzyme intermediate.
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PMID:Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate. 1559 22

beta-Alanine synthase (EC 3.5.1.6) catalyzes the conversion of N-carbamyl-beta-alanine to beta-alanine, ammonia and CO2. The enzyme has been purified to apparent homogeneity from calf liver. The molecular size, pH optimum and substrate specificity have been determined. Sequence alignment of beta-alanine synthases with N-carbamyl-D-amino acid amidohydrolase from Agrobacter sp. revealed the conservation of a catalytically important triad Glu-Lys-Cys, most likely involved in the breakdown of N-carbamyl-beta-alanine.
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PMID:Purification and properties of beta-alanine synthase from calf liver. 1563 4

The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK(a) of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.
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PMID:Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports. 1565 25

The amidase activity of bovine pancreas trypsin in water-soluble complexes with poly(ethylene glycol)-block-poly(alpha,beta-aspartic acid) (PEG-PAA) was evaluated by a colorimetric assay using L-lysine p-nitroanilide as a substrate. The enzymatic reaction of trypsin was accelerated through the complexation with PEG-PAA. By determining the kinetic parameters of the enzymatic reaction of trypsin, it was confirmed that the catalytic rate constant of the complexed trypsin was 15 times higher than that of the native trypsin. From the evaluation of pH dependence of initial reaction rate, it was indicated that this acceleration was induced by a stabilization of the imidazolium ion of the His residue in the catalytic site, the Asp-His-Ser triad, of trypsin due to the Asp units of PEG-PAA. The hydrogen bonded Asp-His pairs are critical constituents in several key enzymatic reactions including serine protease and apurinic endonucleases, and it was expected that the acceleration of the catalytic reaction might occur for other enzymes by the formation of water-soluble complexes with PEG-PAA.
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PMID:Acceleration of enzymatic reaction of trypsin through the formation of water-soluble complexes with poly(ethylene glycol)-block-poly(alpha,beta-aspartic acid). 1576 22

To investigate the role of arginine in the folding of d-aminoacylase, seven arginine residues, R26, R152, R296, R302, R354, R377, and R391, among twelve arginine residues highly conserved in d-aminoacylase, N-acyl-d-aspartate amidohydrolase (d-AAase), and N-acyl-d-glutamate amidohydrolase (d-AGase) from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were substituted with lysine by site-directed mutagenesis. The mutants, R26K, R152K, R296K, and R302K were identified as mutations that increase partitioning of the enzyme into inclusion bodies. No mutants with substitutions within the carboxyterminal segment were found to increase partitioning into inclusion bodies (R354K, R377K, and R392K). These results suggest that arginine residues that position between the N-terminus and central region can play an important role in facilitating folding or stabilizing the structure of d-aminoacylase. By anaerobic cultivation, the production level of R302K in the soluble fraction was improved. Coexpression of the DnaK-DnaJ-GrpE chaperone assisted the folding of R302K, and reduced the effect of the aeration conditions on the solubility of R302K. We hypothesized that R302K requires a larger amount of chaperones for efficient folding than the wild type enzyme.
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PMID:Role of arginine residues of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. 1577 81

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.
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PMID:Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP. 1590 97

We have developed a novel methodology that allowed the preparation of cross-linked enzyme aggregates (CLEAs) of glutaryl acylase (GAC) by co-aggregation of the enzyme with an aminated polymer: polyethyleneimine (PEI). The preparation of CLEAs of GAC from Pseudomonas sp. is not possible when using poly(ethylene glycol) and glutaraldehyde directly as precipitating and cross-linking agent, respectively. This problem arises probably from the low content of surface Lys groups of GAC which prevents an efficient cross-linking of the enzyme molecules in the aggregate. This fact was proven by the release of enzyme molecules from the aggregate and the solubilization of the enzyme when eliminating the precipitating agent. Our new co-aggregation system favors the cross-linking between the very reactive and abundant primary amino groups of the PEI and the primary amino groups on the enzyme surface. The use of PEI prevents the release of enzyme molecules from the aggregate. By this methodology, we prepared a very stable immobilized derivative of GAC. After optimization of the glutaraldehyde treatment conditions, the stability of the enzyme was significantly improved. It kept more than 60% of its initial activity after 72 h of incubation at 45 degrees C, whereas the soluble enzyme was fully inactivated in 2.5 h of incubation in the same conditions. Therefore, we have a new protocol for carrying out the preparation of cross-linked aggregates of enzymes with a low number of lysines on its surface.
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PMID:Co-aggregation of enzymes and polyethyleneimine: a simple method to prepare stable and immobilized derivatives of glutaryl acylase. 1600 17


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