scholarly journals New Fks Hot Spot for Acquired Echinocandin Resistance inSaccharomyces cerevisiaeand Its Contribution to Intrinsic Resistance ofScedosporiumSpecies

2011 ◽  
Vol 55 (8) ◽  
pp. 3774-3781 ◽  
Author(s):  
Michael E. Johnson ◽  
Santosh K. Katiyar ◽  
Thomas D. Edlind
Keyword(s):  
Hot Spot ◽  
Acquired Resistance ◽  
Direct Interaction ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Cell Wall Polysaccharide ◽  
Intrinsic Resistance ◽  
Scedosporium Prolificans ◽  
Echinocandin Resistance

ABSTRACTEchinocandins represent a new antifungal group with potent activity againstCandidaspecies. These lipopeptides inhibit the synthesis of β-1,3-glucan, the major cell wall polysaccharide. Acquired resistance or reduced echinocandin susceptibility (RES) is rare and associated with mutations in two “hot spot” regions of Fks1 or Fks2, the probable β-1,3-glucan synthases. In contrast, many fungi demonstrate intrinsic RES for reasons that remain unclear. We are usingSaccharomyces cerevisiaeto understand the basis for RES by modeling echinocandin-Fks interaction. Previously characterized mutations confer cross-RES; we screened for mutations conferring differential RES, implying direct interaction of that Fks residue with a variable echinocandin side chain. One mutant (in anfks1Δ background) exhibited ≥16-fold micafungin and anidulafungin versus caspofungin RES. Sequencing identified a novel Fks2 mutation, W714L/Y715N. Equivalent W695L/Y696N and related W695L/F/C mutations in Fks1 generated by site-directed mutagenesis and the isolation of a W695L-equivalent mutation inCandida glabrataconfirmed the role of the new “hot spot 3” in RES. Further mutagenesis expanded hot spot 3 to Fks1 residues 690 to 700, yielding phenotypes ranging from cross-RES to differential hypersusceptibility. Fks1 sequences from intrinsically RESScedosporiumspecies revealed W695F-equivalent substitutions; Fks1 hybrids expressingScedosporium prolificanshot spot 3 confirmed that this substitution imparts RES.

scholarly journals Role of embB in natural and acquired resistance to ethambutol in mycobacteria.

1997 ◽  
Vol 41 (10) ◽  
pp. 2270-2273 ◽  
Author(s):  
F Alcaide ◽  
G E Pfyffer ◽  
A Telenti
Keyword(s):  
Nontuberculous Mycobacteria ◽  
Practical Implication ◽  
Acquired Resistance ◽  
Fold Increase ◽  
Drug Susceptibility ◽  
Natural Resistance ◽  
Intrinsic Resistance ◽  
Resistant Strains ◽  
High Level

The mycobacterial embCAB operon encodes arabinosyl transferases, putative targets of the antimycobacterial agent ethambutol (EMB). Mutations in embB lead to resistance to EMB in Mycobacterium tuberculosis. The basis for natural, intrinsic resistance to EMB in nontuberculous mycobacteria (NTM) is not known; neither is the practical implication of resistance to EMB in the absence of embB mutations in M. tuberculosis well understood. The conserved embB resistance-determining region (ERDR) of a collection of 13 strains of NTM and 12 EMB-resistant strains of M. tuberculosis was investigated. Genotypes were correlated with drug susceptibility phenotypes. High-level natural resistance to EMB (MIC, . or =64 microg/ml) was associated with a variant amino acid motif in the ERDR of M. abscessus, M. chelonae, and M. leprae. Transfer of the M. abscessus emb allele to M. smegmatis resulted in a 500-fold increase in the MICs. In M. tuberculosis, embB mutations were associated with MICs of > or =20 microg/ml while resistance not associated with an ERDR mutation generally resulted in MICs of < or =10 microg/ml. These data further support the notion that the emb region determines intrinsic and acquired resistance to EMB and might help in the reassessment of the current recommendations for the screening and treatment of infections with EMB-resistant M. tuberculosis and NTM.

scholarly journals Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

1999 ◽  
Vol 43 (3) ◽  
pp. 543-548 ◽  
Author(s):  
Sonia Trépanier ◽  
James R. Knox ◽  
Natalie Clairoux ◽  
François Sanschagrin ◽  
Roger C. Levesque ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Enterobacter Cloacae ◽  
Acid Group ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Class C ◽  
Three Dimensional Models ◽  
Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

A negatively charged residue in the outer mouth of rat sodium channel determines the gating kinetics of the channel

2003 ◽  
Vol 284 (5) ◽  
pp. C1247-C1254 ◽  
Author(s):  
Zhao Zhang ◽  
Yanfang Xu ◽  
Pei Hong Dong ◽  
Dipika Sharma ◽  
Nipavan Chiamvimonvat
Keyword(s):  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Slow Inactivation ◽  
Voltage Gated ◽  
Charged Residue ◽  
Gating Charge ◽  
Rate Of Recovery ◽  
Domain I ◽  
Pore Lining

Previous studies using combined techniques of site-directed mutagenesis and electrophysiology of voltage-gated Na+ channels have demonstrated that there are significant overlaps in the regions that are important for the two fundamental properties of the channels, namely gating and permeation. We have previously shown that a pore-lining residue, W402 in S5-S6 region (P loop) in domain I of the μ1 skeletal muscle Na+channel, was important in the gating of the channel. Here, we determined the role of an adjacent pore-lining negatively charged residue (E403) in channel gating. Charge neutralization or substitution with positively charged side chain at this position resulted in a marked delay in the rate of recovery from slow inactivation. Indeed, the fast inactivation process appeared intact. Restoration of the negatively charged side chain with a sulfhydryl modifier, MTS-ethylsulfonate, resulted in a reactivation profile from a slow-inactivated state, which was indistinguishable from that of the wild-type channels. We propose an additional functional role for the negatively charged residue. Assuming no major changes in the pore structure induced by the mutations, the negatively charged residue E403 may work in concert with other pore regions during recovery from slow inactivation of the channel. Our data represent the first report indicating the role of negative charge in the slow inactivation of the voltage-gated Na+ channel.

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 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 Rapid Development of Candida krusei Echinocandin Resistance during Caspofungin Therapy

2015 ◽  
Vol 59 (11) ◽  
pp. 6975-6982 ◽  
Author(s):  
A. Forastiero ◽  
V. Garcia-Gil ◽  
O. Rivero-Menendez ◽  
R. Garcia-Rubio ◽  
M. C. Monteiro ◽  
...  
Keyword(s):  
Hot Spot ◽  
Rapid Development ◽  
Lymphocytic Leukemia ◽  
Molecular Evidence ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Rapid Acquisition ◽  
Clinical Resistance ◽  
Echinocandin Resistance

ABSTRACTIn invasive candidiasis, there has been an epidemiological shift fromCandida albicansto non-albicansspecies infections, including infections withC. glabrata,C. parapsilosis,C. tropicalis, andC. krusei. Although the prevalence ofC. kruseiremains low among yeast infections, its intrinsic resistance to fluconazole raises epidemiological and therapeutic concerns. Echinocandins havein vitroactivity against mostCandidaspp. and are the first-line agents in the treatment of candidemia. Although resistance to echinocandin drugs is still rare, individual cases ofC. kruseiresistance have been reported in recent years, especially with strains that have been under selective pressure. A total of 15C. kruseistrains, isolated from the blood, urine, and soft tissue of an acute lymphocytic leukemia patient, were analyzed. Strains developed echinocandin resistance during 10 days of caspofungin therapy. The molecular epidemiology of the isolates was investigated using two different typing methods: PCR-based amplification of the species-specific repetitive polymorphic CKRS-1 sequence and multilocus sequence typing. All isolates were genetically related, and the mechanism involved in decreased echinocandin susceptibility was characterized. Clinical resistance was associated with an increase in echinocandin MICsin vitroand was related to three different mutations in hot spot 1 of the target enzyme Fks1p. Molecular evidence of the rapid acquisition of resistance by different mutations inFKS1highlights the need to monitor the development of resistance inC. kruseiinfections treated with echinocandin drugs.

scholarly journals The role of cysteine residues in the allosteric modulation of the chromophore phototransformations of biphotochromic fluorescent protein SAASoti

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. V. Gavshina ◽  
N. K. Marynich ◽  
M. G. Khrenova ◽  
I. D. Solovyev ◽  
A. P. Savitsky
Keyword(s):  
Fluorescent Protein ◽  
Thermal Relaxation ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Cysteine Residues ◽  
Monomeric Form ◽  
Dynamic Simulations ◽  
High Concentrations ◽  
Mutant Forms

AbstractBiphotochromic fluorescent protein SAASoti contains five cysteine residues in its sequence and a V127T point mutation transforms it to the monomeric form, mSAASoti. These cysteine residues are located far from the chromophore and might control its properties only allosterically. The influence of individual, double and triple cysteine substitutions of mSAASoti on fluorescent parameters and phototransformation reactions (irreversible green-to-red photoconversion and reversible photoswitching) is studied. A set of mSAASoti mutant forms (C21N, C117S, C71V, C105V, C175A, C21N/C71V, C21N/C175A, C21N/C71G/C175A) is obtained by site-directed mutagenesis. We demonstrate that the C21N variant exists in a monomeric form up to high concentrations, the C71V substitution accelerates photoconversion to the red form and the C105V variant has the maximum photoswitching rate. All C175A-containing variants demonstrate different photoswitching kinetics and decreased photostability during subsequent switching cycles compared with other considered systems. Classical molecular dynamic simulations reveal that the F177 side chain located in the vicinity of the chromophore is considerably more flexible in the mSAASoti compared with its C175A variant. This might be the explanation of the experimentally observed slowdown the thermal relaxation rate, i.e., trans–cis isomerization of the chromophore in mSAASoti upon C175A substitution.

scholarly journals Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues

1996 ◽  
Vol 319 (1) ◽  
pp. 315-321 ◽  
Author(s):  
Kian-Leong TAN ◽  
Gareth CHELVANAYAGAM ◽  
Michael W. PARKER ◽  
Philip G. BOARD
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Cumene Hydroperoxide ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Serine Residue ◽  
Subsequent Removal ◽  
Carbonium Ion

The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133–2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247–250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with 1-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of 1-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.

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