Phospholipase A2 engineering. 6. Single amino acid substitutions of active site residues convert the rigid enzyme to highly flexible conformational states

1992 ◽  
Vol 114 (7) ◽  
pp. 2748-2749 ◽  
Author(s):  
Cynthia M. Dupureur ◽  
Yishan Li ◽  
Ming Daw Tsai
Keyword(s):  
Amino Acid ◽  
Phospholipase A2 ◽  
Active Site ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Active Site Residues ◽  
Conformational States

scholarly journals A general method to predict the effect of single amino acid substitutions on enzyme catalytic activity

2017 ◽  
Author(s):  
Yu-Hsiu T. Lin ◽  
Cheng Lai Victor Huang ◽  
Christina Ho ◽  
Max Shatsky ◽  
Jack F. Kirsch
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Active Site ◽  
Enzyme Reaction ◽  
Site Directed Mutagenesis ◽  
Enzyme Function ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Amino Acid Substitutions ◽  
Active Site Residues

ABSTRACTOver the past thirty years, site-directed mutagenesis has become established as one of the most powerful techniques to probe enzyme reaction mechanisms1-3. Substitutions of active site residues are most likely to yield significant perturbations in kinetic parameters, but there are many examples of profound changes in these values elicited by remote mutations4-6. Ortholog comparisons of extant sequences show that many mutations do not have profound influence on enzyme function. As the number of potential single natural amino acid substitutions that can be introduced in a protein of N amino acids in length by directed mutation is very large (19 * N), it would be useful to have a method to predict which amino acid substitutions are more likely to introduce significant changes in kinetic parameters in order to design meaningful probes into enzyme function. What is especially desirable is the identification of critical residues that do not contact the substrate directly, and may be remote from the active site.We collected literature data reflecting the effects of 2,804 mutations on kinetic properties for 12 enzymes. These data along with characteristic predictors were used in a machine-learning scheme to train a classifier to predict the effect of mutation. Use of this algorithm allows one to predict with a 2.5-fold increase in precision, if a given mutation, made anywhere in the enzyme, will cause a decrease in kcat/Km value of ≥ 95%. The improved precision allows the experimentalist to reduce the number of mutations necessary to probe the enzyme reaction mechanism.

scholarly journals A Critical Role of Non-active Site Residues on Cyclooxygenase Helices 5 and 6 in the Control of Prostaglandin Stereochemistry at Carbon 15

2007 ◽  
Vol 282 (38) ◽  
pp. 28157-28163 ◽  
Author(s):  
Karin Valmsen ◽  
William E. Boeglin ◽  
Reet Järving ◽  
Ivar Järving ◽  
Külliki Varvas ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Amino Acid Sequence Identity ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Active Site Residues ◽  
The Core ◽  
The Difference

The correct stereochemistry of prostaglandins is a prerequisite of their biological activity and thus is under a strict enzymatic control. Recently, we cloned and characterized two cyclooxygenase (COX) isoforms in the coral Plexaura homomalla that share 97% amino acid sequence identity, yet form prostaglandins with opposite stereochemistry at carbon 15. The difference in oxygenation specificity is only partially accounted for by the single amino acid substitution in the active site (Ile or Val at position 349). For further elucidation of residues involved in the C-15 stereocontrol, a series of sequence swapping and site-directed mutagenesis experiments between 15R- and 15S-COX were performed. Our results show that the change in stereochemistry at carbon 15 of prostaglandins relates mainly to five amino acid substitutions on helices 5 and 6 of the coral COX. In COX proteins, these helices form a helix-turn-helix motif that traverses through the entire protein, contributing to the second shell of residues around the oxygenase active site; it constitutes the most highly conserved region where even slight changes result in loss of catalytic activity. The finding that this region is among the least conserved between the P. homomalla 15S- and 15R-specific COX further supports its significance in maintaining the desired prostaglandin stereochemistry at C-15. The results are particularly remarkable because, based on its strong conservation, the conserved middle of helix 5 is considered as central to the core structure of peroxidases, of which COX proteins are derivatives. Now we show that the same parts of the protein are involved in the control of oxygenation with 15R or 15S stereospecificity in the dioxygenase active site.

scholarly journals Characterization of the AmpC β-Lactamase fromBurkholderia multivorans

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Scott A. Becka ◽  
Elise T. Zeiser ◽  
Melissa D. Barnes ◽  
Magdalena A. Taracila ◽  
Kevin Nguyen ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Active Site ◽  
Burkholderia Cepacia ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Enzyme Complex ◽  
Active Site Residues ◽  
Content Type ◽  
Burkholderia Multivorans

ABSTRACTBurkholderia multivoransis a member of theBurkholderia cepaciacomplex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. β-Lactam antibiotics are recommended as therapy for infections due toB.multivorans, which possesses two β-lactamase genes,blapenAandblaAmpC. PenA is a carbapenemase with a substrate profile similar to that of theKlebsiella pneumoniaecarbapenemase (KPC); in addition, expression of PenA is inducible by β-lactams inB.multivorans. Here, we characterize AmpC fromB.multivoransATCC 17616. AmpC possesses only 38 to 46% protein identity with non-BurkholderiaAmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates ofB.multivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Ω loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward β-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (Kiapp> 600 μM), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible inB.multivoransATCC 17616 after growth in subinhibitory concentrations of imipenem (1 μg/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role inB.multivoranscompared to PenA, which is the dominant β-lactamase inB.multivoransATCC 17616.

scholarly journals Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

2000 ◽  
Vol 182 (6) ◽  
pp. 1641-1649 ◽  
Author(s):  
Rebecca E. Parales ◽  
Kyoung Lee ◽  
Sol M. Resnick ◽  
Haiyan Jiang ◽  
Daniel J. Lessner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Product Formation ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Substitutions ◽  
Active Site Residues ◽  
Naphthalene Dioxygenase ◽  
Wide Range

ABSTRACT The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene byPseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation ofcis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.

scholarly journals Mutational Analysis of the Escherichia coli melR Gene Suggests a Two-State Concerted Model To Explain Transcriptional Activation and Repression in the Melibiose Operon

2006 ◽  
Vol 188 (9) ◽  
pp. 3199-3207 ◽  
Author(s):  
Christina Kahramanoglou ◽  
Christine L. Webster ◽  
Mohamed Samir el-Robh ◽  
Tamara A. Belyaeva ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Hybrid System ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Supporting Evidence ◽  
Conformational States ◽  
Two Hybrid

ABSTRACT Transcription of the Escherichia coli melAB operon is regulated by the MelR protein, an AraC family member whose activity is modulated by the binding of melibiose. In the absence of melibiose, MelR is unable to activate the melAB promoter but autoregulates its own expression by repressing the melR promoter. Melibiose triggers MelR-dependent activation of the melAB promoter and relieves MelR-dependent repression of the melR promoter. Twenty-nine single amino acid substitutions in MelR that result in partial melibiose-independent activation of the melAB promoter have been identified. Combinations of different substitutions result in almost complete melibiose-independent activation of the melAB promoter. MelR carrying each of the single substitutions is less able to repress the melR promoter, while MelR carrying some combinations of substitutions is completely unable to repress the melR promoter. These results argue that different conformational states of MelR are responsible for activation of the melAB promoter and repression of the melR promoter. Supporting evidence for this is provided by the isolation of substitutions in MelR that block melibiose-dependent activation of the melAB promoter while not changing melibiose-independent repression of the melR promoter. Additional experiments with a bacterial two-hybrid system suggest that interactions between MelR subunits differ according to the two conformational states.

scholarly journals Modulation of Enzymatic Activity and Biological Function of Listeria monocytogenes Broad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with theBacillus cereus Ortholog

1998 ◽  
Vol 66 (10) ◽  
pp. 4823-4831 ◽  
Author(s):  
Wolfram R. Zückert ◽  
Hélène Marquis ◽  
Howard Goldfine
Keyword(s):  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Phospholipase C ◽  
Active Site ◽  
Mammalian Cells ◽  
Biological Function ◽  
Single Amino Acid ◽  
Broth Culture ◽  
Amino Acid Substitutions ◽  
Wild Type

ABSTRACT The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs,Clostridium perfringens α-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted inL. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

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