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 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 Albino mutants of Streptomyces glaucescens tyrosinase

1991 ◽  
Vol 274 (3) ◽  
pp. 707-713 ◽  
Author(s):  
M P Jackman ◽  
A Hajnal ◽  
K Lerch
Keyword(s):  
Active Site ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Carbonyl Derivatives ◽  
Hydrogen Bonding Interactions ◽  
Albino Phenotype ◽  
Derivatives Of ◽  
Streptomyces Glaucescens

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis

Biochimie ◽  
2007 ◽  
Vol 89 (12) ◽  
pp. 1498-1508 ◽  
Author(s):  
Muriel Crouvoisier ◽  
Geneviève Auger ◽  
Didier Blanot ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals A sensitive core region in the structure of glutathione S-transferases

2003 ◽  
Vol 373 (3) ◽  
pp. 759-765 ◽  
Author(s):  
Jantana WONGSANTICHON ◽  
Thasaneeya HARNNOI ◽  
Albert J. KETTERMAN
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Single Amino Acid ◽  
Variant Form ◽  
Active Site Residues ◽  
Glutathione S Transferase ◽  
Anopheles Dirus ◽  
Single Amino Acid Change ◽  
Exclusion Chromatography

A variant form of an Anopheles dirus glutathione S-transferase (GST), designated AdGSTD4-4, possesses a single amino acid change of leucine to arginine (Leu-103-Arg). Although residue 103 is outside of the active site, it has major effects on enzymic properties. To investigate these structural effects, site-directed mutagenesis was used to generate mutants by changing the non-polar leucine to alanine, glutamate, isoleucine, methionine, asparagine, or tyrosine. All of the recombinant GSTs showed approximately the same expression level at 25° C. Several of the mutants lacked glutathione (GSH)-binding affinity but were purified by S-hexyl-GSH-based affinity chromatography. However the protein yields (70-fold lower), as well as the GST activity (100-fold lower), of Leu-103-Tyr and Leu-103-Arg purifications were surprisingly low and precluded the performance of kinetic experiments. Size-exclusion chromatography showed that both GSTs Leu-103-Tyr and Leu-103-Arg formed dimers. Using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH substrates to determine kinetic constants it was demonstrated that the other Leu-103 mutants possessed a greater Km towards GSH and a differing Km towards CDNB. The Vmax ranged from 44.7 to 87.0 μmol/min per mg (wild-type, 44.7 μmol/min per mg). Substrate-specificity studies showed different selectivity properties for each mutant. The structural residue Leu-103 affects the active site through H-bond and van-der-Waal contacts with six active-site residues in the GSH binding site. Changes in this interior core residue appear to disrupt internal packing, which affects active-site residues as well as residues at the subunit–subunit interface. Finally, the data suggest that Leu-103 is noteworthy as a sensitive residue in the GST structure that modulates enzyme activity as well as stability.

scholarly journals Efficient Isolation of Wild Strains of Canine Distemper Virus in Vero Cells Expressing Canine SLAM (CD150) and Their Adaptability to Marmoset B95a Cells

2003 ◽  
Vol 77 (18) ◽  
pp. 9943-9950 ◽  
Author(s):  
Fumio Seki ◽  
Nobuyuki Ono ◽  
Ryoji Yamaguchi ◽  
Yusuke Yanagi
Keyword(s):  
Amino Acid ◽  
Canine Distemper Virus ◽  
Lymphocyte Activation ◽  
Vero Cells ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Canine Distemper ◽  
Wild Strains ◽  
The Difference ◽  
Fusion Analysis

ABSTRACT We have previously shown that canine signaling lymphocyte activation molecule (SLAM; also known as CD150) acts as a cellular receptor for canine distemper virus (CDV). In this study, we established Vero cells stably expressing canine SLAM (Vero.DogSLAMtag cells). Viruses were isolated in Vero.DogSLAMtag cells one day after inoculation with spleen samples from five out of seven dogs with distemper. By contrast, virus isolation with reportedly sensitive marmoset B95a cells was only successful from three diseased animals at 7 to 10 days after inoculation, and no virus was recovered from any dogs when Vero cells were used for isolation. The CDV strain isolated in Vero.DogSLAMtag cells did not cause cytopathic effects in B95a and human SLAM-expressing Vero cells, whereas the strain isolated in B95a cells from the same dog did so in canine or human SLAM-expressing Vero cells as well as B95a cells. There were two amino acid differences in the hemagglutinin sequence between these strains. Cell fusion analysis after expression of envelope proteins and vesicular stomatitis virus pseudotype assay showed that their hemagglutinins were responsible for the difference in cell tropism between them. Site-directed mutagenesis indicated that glutamic acid to lysine substitution at position 530 of the hemagglutinin was required for the adaptation to the usage of marmoset SLAM. Our results indicate that Vero cells stably expressing canine SLAM are highly sensitive to CDV in clinical specimens and that only a single amino acid substitution in the hemagglutinin can allow the virus to adapt to marmoset SLAM.

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 Role of Arginine 214 in the Substrate Specificity of OXA-48

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Saoussen Oueslati ◽  
Pascal Retailleau ◽  
Ludovic Marchini ◽  
Camille Berthault ◽  
Laurent Dortet ◽  
...  
Keyword(s):  
Amino Acid ◽  
Salt Bridge ◽  
Amino Acid Position ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Nucleophilic Attack ◽  
Kinetic Properties ◽  
X Ray

ABSTRACT Increasing numbers of variants of the carbapenem-hydrolyzing class D β-lactamase OXA-48 are identified in Enterobacterales worldwide. Among them, OXA-181 and OXA-232 are of particular interest, as they differ from each other by a single amino acid substitution at position 214 (R in OXA-181 and S in OXA-232) that results in reduced carbapenem-hydrolyzing activity for OXA-232. To investigate the role of amino acid position 214 (AA214), the X-ray structure of OXA-232 was determined and AA214 of OXA-48 and of OXA-232 was replaced by G, L, D, E, S, R, and K using site-directed mutagenesis. These mutants were phenotypically characterized, and three mutants of OXA-232 were purified to study their steady-state kinetic properties. The X-ray structure of OXA-232 along with molecular modeling studies showed that the interaction via a salt bridge between R214 and D159 in OXA-48 is not possible with the G214 or S214 mutation. In contrast, with K214, which is also positively charged, the interaction with D159 is maintained. With the E214 mutant, an alternative binding conformation of imipenem that is not compatible with a nucleophilic attack by S70 was evidenced. Thus, imipenem has a very poor apparent affinity for the E214 mutant because of its nonproductive binding mode. Similarly, we could explain the lack of temocillin hydrolysis by the OXA-232-S214E mutant, which is due to the unfavorable interaction between the negatively charged R1 substituent of temocillin with the E214 residue. Overall, we demonstrate that AA214 in OXA-48-like β-lactamases is critical for the carbapenemase activity.

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