scholarly journals Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-l-alanine amidase

2004 ◽  
Vol 377 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Catherine GÉNÉREUX ◽  
Dominique DEHARENG ◽  
Bart DEVREESE ◽  
Jozef VAN BEEUMEN ◽  
Jean-Marie FRÈRE ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Hydrolytic Enzymes ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Activity

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-l-alanine amidase involved in both peptidoglycan recycling and β-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.

scholarly journals Crystal Structure of an Iron-Dependent Group III Dehydrogenase That Interconverts l-Lactaldehyde and l-1,2-Propanediol in Escherichia coli

2005 ◽  
Vol 187 (14) ◽  
pp. 4957-4966 ◽  
Author(s):  
Cristina Montella ◽  
Lluis Bellsolell ◽  
Rosa Pérez-Luque ◽  
Josefa Badía ◽  
Laura Baldoma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Protein A ◽  
Close Approach ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Group Iii ◽  
Terminal Domain ◽  
Α Helix

ABSTRACT The FucO protein, a member of the group III “iron-activated” dehydrogenases, catalyzes the interconversion between l-lactaldehyde and l-1,2-propanediol in Escherichia coli. The three-dimensional structure of FucO in a complex with NAD+ was solved, and the presence of iron in the crystals was confirmed by X-ray fluorescence. The FucO structure presented here is the first structure for a member of the group III bacterial dehydrogenases shown experimentally to contain iron. FucO forms a dimer, in which each monomer folds into an α/β dinucleotide-binding N-terminal domain and an all-α-helix C-terminal domain that are separated by a deep cleft. The dimer is formed by the swapping (between monomers) of the first chain of the β-sheet. The binding site for Fe2+ is located at the face of the cleft formed by the C-terminal domain, where the metal ion is tetrahedrally coordinated by three histidine residues (His200, His263, and His277) and an aspartate residue (Asp196). The glycine-rich turn formed by residues 96 to 98 and the following α-helix is part of the NAD+ recognition locus common in dehydrogenases. Site-directed mutagenesis and enzyme kinetic assays were performed to assess the role of different residues in metal, cofactor, and substrate binding. In contrast to previous assumptions, the essential His267 residue does not interact with the metal ion. Asp39 appears to be the key residue for discriminating against NADP+. Modeling l-1,2-propanediol in the active center resulted in a close approach of the C-1 hydroxyl of the substrate to C-4 of the nicotinamide ring, implying that there is a typical metal-dependent dehydrogenation catalytic mechanism.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Proteolytic Activity ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

2007 ◽  
Vol 405 (3) ◽  
pp. 445-454 ◽  
Author(s):  
Tanja Schlecker ◽  
Marcelo A. Comini ◽  
Johannes Melchers ◽  
Thomas Ruppert ◽  
R. Luise Krauth-Siegel
Keyword(s):  
Glutathione Peroxidase ◽  
Trypanosoma Brucei ◽  
Catalytic Mechanism ◽  
Disulfide Bridge ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Glutathione Peroxidases ◽  
Thiolate Anion ◽  
In The Wild

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.

scholarly journals Mutational analysis of the Drosophila snake protease: an essential role for domains within the proenzyme polypeptide chain.

Genetics ◽  
1994 ◽  
Vol 136 (4) ◽  
pp. 1355-1365 ◽  
Author(s):  
C Smith ◽  
H Giordano ◽  
R DeLotto
Keyword(s):  
Serine Proteases ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Normal Activity ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Rna Transcripts ◽  
Intron 1 ◽  
Regulation Of Activity

Abstract Two genes involved in the generation of dorsoventral asymmetry in the developing Drosophila melanogaster embryo, snake and easter, encode the zymogen form of serine proteases. Mutant alleles of snake were cloned and sequenced revealing two types of lesions: point mutations which alter the amino acid sequence (snk073 and snkrm4) and point mutations which alter the splicing (snk229 or snk233) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site. snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA. RNA phenotypic rescue and site-directed mutagenesis experiments indicate that snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity. Deletion experiments show that an acidic proenzyme domain is required for snake rescue activity to be uniformly distributed throughout the embryo. A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of activity of the snake catalytic chain. Transcripts encoding only the proenzyme polypeptides of either snake or easter can dorsalize wild type embryos. We propose a model in which the proenzyme determinants of both the snake and easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system.

Mutational analysis of transmembrane histidines in the amiloride-sensitive Na+/H+ exchanger

1995 ◽  
Vol 269 (2) ◽  
pp. C392-C402 ◽  
Author(s):  
D. Wang ◽  
D. F. Balkovetz ◽  
D. G. Warnock
Keyword(s):  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
Clear Cut ◽  
Inhibition Concentration ◽  
Nhe1 Activity ◽  
Per Se ◽  
Nhe Isoforms

The histidine-reactive reagent, diethyl pyrocarbonate (DEPC) inhibits the human amiloride-sensitive Na+/H+ exchanger (NHE1) in stably transfected fibroblasts. NHE1 was protected by cimetidine and amiloride from DEPC, and DEPC inhibition was reversed with hydroxylamine, suggesting a role for critical histidine groups in NHE activity. We replaced the histidines (H) in putative transmembrane domains (H35, H120, H349) with glycine (G) using site-directed mutagenesis. There was no significant change in NHE activity of the H120G; H349G; H120,349G; and H35,120,349G mutants compared with wild type. The 50% inhibition concentration values for amiloride, ethyl isopropyl amiloride (EIPA), and cimetidine of the H349G mutant were significantly increased compared with the wild-type NHE1. We also examined the DEPC effect on the transport activity of the triple histidine mutant (H35,120,349G) and found that NHE1 activity was still inhibited by DEPC with reversal by hydroxylamine and protected by amiloride and cimetidine. Kinetic analysis of DEPC inhibition indicated that two "critical" histidine residues are required for NHE transport activity. Substitutions of H349 with asparagine (N), glutamine (Q), serine (S), tyrosine (Y), valine (V), leucine (L), and phenylalanine (F) were also examined. There were no changes in NHE activity of these mutants compared with wild type. The H349G and H349L mutants became more resistant to amiloride, whereas the H349Y and H349F mutants became more sensitive to amiloride. The H349S (mimics NHE3) and H349Y (mimics NHE4) mutations had only modest effects on amiloride sensitivity. These results indicate that H349 affects the interaction of NHE1 with its inhibitors, even though substitutions at this site, per se, do not appear to explain the differences in amiloride sensitivity between different NHE isoforms. Despite clear-cut effects of the H349G mutation on the competitive interaction of NHE1 with cimetidine and EIPA, this mutation did not affect the affinity of NHE1 for its cationic substrates (Na+, Li+).

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia Pastoris

2020 ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Km Value ◽  
Autolytic Activity

Abstract Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.

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