scholarly journals Alterations in PBP 1A Essential for High-Level Penicillin Resistance in Streptococcus pneumoniae

1998 ◽  
Vol 42 (6) ◽  
pp. 1329-1333 ◽  
Author(s):  
Anthony M. Smith ◽  
Keith P. Klugman
Keyword(s):  
Amino Acid ◽  
South African ◽  
Site Directed Mutagenesis ◽  
Penicillin Resistance ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Penicillin Binding Proteins ◽  
Development Of Resistance ◽  
High Level

ABSTRACT High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 μg/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 μg/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 μg/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 μg/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 μg/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 μg/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.

scholarly journals Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

scholarly journals Mechanism of Darunavir (DRV)’s High Genetic Barrier to HIV-1 Resistance: A Key V32I Substitution in Protease Rarely Occurs, but Once It Occurs, It Predisposes HIV-1 To Develop DRV Resistance

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Manabu Aoki ◽  
Debananda Das ◽  
Hironori Hayashi ◽  
Hiromi Aoki-Ogata ◽  
Yuki Takamatsu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Critical Role ◽  
Viral Fitness ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Genetic Barrier ◽  
High Level ◽  
Hiv 1

ABSTRACTDarunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIVDRVRP51). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIVDRVRP51are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIVI54Mand rHIVI84V, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIVV32Ialso developed high-level DRV resistance. However, wild-type HIVNL4-3(rHIVWT) failed to acquire V32I and did not develop DRV resistance. Compared to rHIVWT, rHIVV32Iwas highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIVWTwith DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1’s DRV resistance development and elucidate, at least in part, a mechanism of DRV’s high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution.IMPORTANCEDarunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1’s drug resistance. However, the mechanism(s) of the DRV’s high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, allin vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIVWT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIVWT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1’s resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

scholarly journals Alteration of Chain Length Substrate Specificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis

2003 ◽  
Vol 69 (8) ◽  
pp. 4830-4836 ◽  
Author(s):  
Takeharu Tsuge ◽  
Tamao Hisano ◽  
Seiichi Taguchi ◽  
Yoshiharu Doi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Chain Length ◽  
Acyl Chain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Acyl Chain Length ◽  
Aeromonas Caviae

ABSTRACT Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJAc revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1 Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

scholarly journals Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase

1998 ◽  
Vol 330 (2) ◽  
pp. 615-621 ◽  
Author(s):  
Stefano CAMPANER ◽  
Loredano POLLEGIONI ◽  
D. Brian ROSS ◽  
S. Mirella PILONE
Keyword(s):  
Amino Acid ◽  
Isoelectric Focusing ◽  
Limited Proteolysis ◽  
Single Band ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

When analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilisd-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of ≈ 34 kDa (Met1-Leu312) and one of ≈ 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilisD-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers.

scholarly journals VIM-19, a Metallo-β-Lactamase with Increased Carbapenemase Activity from Escherichia coli and Klebsiella pneumoniae

2009 ◽  
Vol 54 (1) ◽  
pp. 471-476 ◽  
Author(s):  
Jose-Manuel Rodriguez-Martinez ◽  
Patrice Nordmann ◽  
Nicolas Fortineau ◽  
Laurent Poirel
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Class 1 Integron ◽  
Carbapenem Resistant ◽  
Class 1

ABSTRACT Two carbapenem-resistant isolates, one Escherichia coli isolate and one Klebsiella pneumoniae isolate, recovered from an Algerian patient expressed a novel VIM-type metallo-β-lactamase (MBL). The identified bla VIM-19 gene was located on a ca. 160-kb plasmid and located inside a class 1 integron in both isolates. VIM-19 differed from VIM-1 by the Asn215Lys and Ser228Arg substitutions, increasing its hydrolytic activity toward carbapenems. Site-directed mutagenesis experiments showed that both substitutions were necessary for the increased carbapenemase activity of VIM-19. This study indicates that MBLs with enhanced activity toward carbapenems may be obtained as a result of very few amino acid substitutions.

Investigating the active site of human trimethyllysine hydroxylase

2019 ◽  
Vol 476 (7) ◽  
pp. 1109-1119 ◽  
Author(s):  
Yali Wang ◽  
Y. Vijayendar Reddy ◽  
Abbas H. K. Al Temimi ◽  
Hanka Venselaar ◽  
Frank H. T. Nelissen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Biosynthesis Pathway ◽  
Recognition Sites ◽  
Aromatic Cage ◽  
Basic Insight ◽  
Insight Into

Abstract The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate and (2S)-Nε-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2S)-Nε-trimethyllysine to (2S,3S)-3-hydroxy-Nε-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242–D244–H389 residues, R391–R398 involved in 2OG binding and several residues (D231, N334 and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2S)-Nε-trimethyllysine.

scholarly journals Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

2019 ◽  
Vol 20 (24) ◽  
pp. 6224 ◽  
Author(s):  
Bangqiao Yin ◽  
Qinyan Hui ◽  
Muhammad Kashif ◽  
Ran Yu ◽  
Si Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Directed Evolution ◽  
Catalytic Efficiency ◽  
Raw Material ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM−1 s−1) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

scholarly journals A Novel FexA Variant from a Canine Staphylococcus pseudintermedius Isolate That Does Not Confer Florfenicol Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5763-5766 ◽  
Author(s):  
Elena Gómez-Sanz ◽  
Kristina Kadlec ◽  
Andrea T. Feßler ◽  
Myriam Zarazaga ◽  
Carmen Torres ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Base Pairs ◽  
Chloramphenicol Resistance ◽  
Content Type ◽  
Resistance Phenotype ◽  
First Time

ABSTRACTTransposon Tn558integrated in the chromosomalradCgene was detected for the first time inStaphylococus pseudintermedius. It carried a novelfexAvariant (fexAv) that confers only chloramphenicol resistance. The exporter FexAv exhibited two amino acid substitutions, Gly33Ala and Ala37Val, both of which seem to be important for substrate recognition. Site-directed mutagenesis that reverted the mutated base pairs to those present in the originalfexAgene restored the chloramphenicol-plus-florfenicol resistance phenotype.

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