scholarly journals The role of the histidine-35 residue in the cytocidal action of HM-1 killer toxin

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2951-2958 ◽  
Author(s):  
Masahiko Miyamoto ◽  
Naohiko Onozato ◽  
Dakshnamurthy Selvakumar ◽  
Tetsuya Kimura ◽  
Yasuhiro Furuichi ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Killer Toxin ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Glucan Synthase ◽  
Killing Activity

Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S. cerevisiae BJ1824 rhk1Δ : : URA3 and HM-1-insensitive yeast Candida albicans even after 1 h incubation. H35A HM-1 inhibited the activity of partially purified 1,3-β-glucan synthase from S. cerevisiae A451, and its extent was almost the same as wild-type HM-1. Co-immunoprecipitation experiments showed that wild-type and H35A HM-1 directly interact with the 1,3-β-glucan synthase complex. These results strongly suggest that histidine-35 has an important role in the cytocidal action of HM-1 that participates in the binding process to the HM-1 receptor protein on the cell membrane, but it is not essential for the interaction with, and inhibition of, 1,3-β-glucan synthase.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

scholarly journals Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

2005 ◽  
Vol 71 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Chan K. Chan Kwo Chion ◽  
Sarah E. Askew ◽  
David J. Leak
Keyword(s):  
Mutational Analysis ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Styrene Oxidation ◽  
Overlapping Reading Frames ◽  
Reading Frames

ABSTRACT Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Alteration of Leucine Aminopeptidase from Streptomyces septatus TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis

2005 ◽  
Vol 71 (11) ◽  
pp. 7229-7235 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Methyl Ester ◽  
Leucine Aminopeptidase ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Peptide Substrates ◽  
Hydrolytic Activities

ABSTRACT To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed l-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with l-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.

scholarly journals Point mutations of two arginine residues in the Streptomyces R61 dd-peptidase

1992 ◽  
Vol 283 (1) ◽  
pp. 123-128 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Ghuysen ◽  
J M Frère
Keyword(s):  
Enzyme Activity ◽  
Enzyme Stability ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Arginine Residues

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

scholarly journals Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

2000 ◽  
Vol 350 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Ana M. PAJOR ◽  
Esther S. KAHN ◽  
Rama GANGULA
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
In The Wild ◽  
Cationic Amino Acids

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

scholarly journals Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine.

1996 ◽  
Vol 40 (9) ◽  
pp. 1983-1987 ◽  
Author(s):  
Y Q Zhu ◽  
K M Remington ◽  
T W North
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Cultured Cells ◽  
Single Point ◽  
Site Directed Mutagenesis ◽  
Single Point Mutation ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Phosphonoformic Acid ◽  
Immunodeficiency Virus

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis.

scholarly journals Topology of the Erwinia chrysanthemi oligogalacturonate porin KdgM

2003 ◽  
Vol 372 (2) ◽  
pp. 329-334 ◽  
Author(s):  
Teijo PELLINEN ◽  
Helena AHLFORS ◽  
Nicolas BLOT ◽  
Guy CONDEMINE
Keyword(s):  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Transmembrane Segments

The Erwinia chrysanthemi oligogalacturonate-specific monomeric porin, KdgM, does not present homology with any porins of known structure. A model of this protein, based on sequence similarity and the amphipathy profile, was constructed. The model depicts a β-barrel composed of 14 antiparallel β-strands. The accuracy of this model was tested by the chemical labelling of cysteine residues introduced by site-directed mutagenesis. The protein has seven surface-exposed loops. They are rather small with the exception of one, loop L6. Deletion of this loop allowed the entry of maltopentaose into the bacteria, a molecule too large to enter through the wild-type KdgM. Loop L6 could fold back into the lumen of the pore and play the role of the constriction loop L3 of general porins. With 14 transmembrane segments, the KdgM porin family could represent the smallest porin characterized to date.

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