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

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.

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Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

Biochemical Journal ◽

10.1042/bj3020355 ◽

1994 ◽

Vol 302 (2) ◽

pp. 355-361 ◽

Cited By ~ 31

Author(s):

K Inukai ◽

T Asano ◽

H Katagiri ◽

M Anai ◽

M Funaki ◽

...

Keyword(s):

Glucose Transporter ◽

Cytochalasin B ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Site Directed Mutagenesis ◽

Transport Activity ◽

Wild Type ◽

In The Wild ◽

Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

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A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct

Biochemical Journal ◽

10.1042/bj3260861 ◽

1997 ◽

Vol 326 (3) ◽

pp. 861-866 ◽

Cited By ~ 14

Author(s):

Timothy P. O'CONNELL ◽

Regina M. DAY ◽

Ekaterina V. TORCHILIN ◽

William W. BACHOVCHIN ◽

J. Paul G. MALTHOUSE

Keyword(s):

13C Nmr ◽

Chemical Shifts ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Imidazolium Cation ◽

Nmr Study ◽

In The Wild ◽

Main Factor

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3–103.6 p.p.m., depending on the pH. The titration shift of 4.7–4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.

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An Unusual Response Regulator Influences Sporulation at Early and Late Stages in Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.01615-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2873-2885 ◽

Cited By ~ 24

Author(s):

Yuqing Tian ◽

Kay Fowler ◽

Kim Findlay ◽

Huarong Tan ◽

Keith F. Chater

Keyword(s):

Streptomyces Coelicolor ◽

Point Mutations ◽

Aerial Mycelium ◽

Site Directed Mutagenesis ◽

Null Mutant ◽

Wild Type ◽

New Variant ◽

In The Wild ◽

Pcr Targeting ◽

Spore Maturation

ABSTRACT WhiI, a regulator required for efficient sporulation septation in the aerial mycelium of Streptomyces coelicolor, resembles response regulators of bacterial two-component systems but lacks some conserved features of typical phosphorylation pockets. Four amino acids of the abnormal “phosphorylation pocket” were changed by site-directed mutagenesis. Unlike whiI null mutations, these point mutations did not interfere with sporulation septation but had various effects on spore maturation. Transcriptome analysis was used to compare gene expression in the wild-type strain, a D27A mutant (pale gray spores), a D69E mutant (wild-type spores), and a null mutant (white aerial mycelium, no spores) (a new variant of PCR targeting was used to introduce the point mutations into the chromosomal copy of whiI). The results revealed 45 genes that were affected by the deletion of whiI. Many of these showed increased expression in the wild type at the time when aerial growth and development were taking place. About half of them showed reduced expression in the null mutant, and about half showed increased expression. Some, but not all, of these 45 genes were also affected by the D27A mutation, and a few were affected by the D69E mutation. The results were consistent with a model in which WhiI acts differently at sequential stages of development. Consideration of the functions of whiI-influenced genes provides some insights into the physiology of aerial hyphae. Mutation of seven whiI-influenced genes revealed that three of them play roles in spore maturation.

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Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00178-09 ◽

2009 ◽

Vol 8 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 13

Author(s):

Maria Lucia Sampaio Güther ◽

Kenneth Beattie ◽

Douglas J. Lamont ◽

John James ◽

Alan R. Prescott ◽

...

Keyword(s):

Cell Surface ◽

Trypanosoma Brucei ◽

Gel Filtration ◽

Apparent Molecular Weight ◽

Life Cycle Stage ◽

Galactose Oxidase ◽

Surface Coat ◽

Wild Type ◽

In The Wild ◽

P Type

ABSTRACT A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB3H4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB3H4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.

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Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1619659114 ◽

2016 ◽

Vol 114 (2) ◽

pp. E132-E141 ◽

Cited By ~ 27

Author(s):

Thiago G. P. Alegria ◽

Diogo A. Meireles ◽

José R. R. Cussiol ◽

Martín Hugo ◽

Madia Trujillo ◽

...

Keyword(s):

Fatty Acid ◽

Wild Type Strain ◽

Wild Type ◽

Glutathione Peroxidases ◽

Mutant Strains ◽

In The Wild ◽

Acid Hydroperoxide ◽

Lipoamide Dehydrogenase ◽

Fatty Acid Hydroperoxides ◽

Competition Assays

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr fromXylella fastidiosaand were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase–coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107–108M−1⋅s−1range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107M−1⋅s−1at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohrmutant ofPseudomonas aeruginosatoward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohrstrain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohrmutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host–pathogen interactions.

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Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

Biochemical Journal ◽

10.1042/bj3020095 ◽

1994 ◽

Vol 302 (1) ◽

pp. 95-101 ◽

Cited By ~ 11

Author(s):

A Schejter ◽

T I Koshy ◽

T L Luntz ◽

R Sanishvili ◽

I Vig ◽

...

Keyword(s):

Cytochrome C ◽

Site Directed Mutagenesis ◽

Wild Type ◽

General Increase ◽

Reduction Potentials ◽

Cytochromes C ◽

In The Wild ◽

Order Of Magnitude ◽

The Stability ◽

Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

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The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

Biochemical Journal ◽

10.1042/bj3120273 ◽

1995 ◽

Vol 312 (1) ◽

pp. 273-280 ◽

Cited By ~ 40

Author(s):

M Haraguchi ◽

S Yamashiro ◽

K Furukawa ◽

K Takamiya ◽

H Shiku ◽

...

Keyword(s):

Enzyme Activity ◽

Intracellular Localization ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Kinetics Analysis ◽

Glycosylation Sites ◽

In The Wild ◽

Polymerase Chain ◽

The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

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

Biochemical Journal ◽

10.1042/bj3030357 ◽

1994 ◽

Vol 303 (2) ◽

pp. 357-362 ◽

Cited By ~ 29

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.

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

Biochemical Journal ◽

10.1042/bj20011468 ◽

2002 ◽

Vol 365 (1) ◽

pp. 303-309 ◽

Cited By ~ 20

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.

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

Genetics ◽

10.1093/genetics/136.4.1355 ◽

1994 ◽

Vol 136 (4) ◽

pp. 1355-1365 ◽

Cited By ~ 6

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.

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