scholarly journals Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

2007 ◽  
Vol 189 (14) ◽  
pp. 5265-5275 ◽  
Author(s):  
Dylan Dodd ◽  
Joseph G. Reese ◽  
Craig R. Louer ◽  
Jimmy D. Ballard ◽  
M. Ashley Spies ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Active Site ◽  
Genomic Sequence ◽  
Ph Dependence ◽  
Size Exclusion ◽  
Kinetic Properties ◽  
Active Site Residues ◽  
Glutamate Racemase ◽  
Genes Encoding ◽  
Exclusion Chromatography

ABSTRACT Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because l-glutamate stereoisomerization to d-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of l-glutamate and d-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of l-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features.

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.

Drugs Against Mycobacterium tuberculosis 3-Isopropylmalate Dehydrogenase Can be Developed Using Homologous Enzymes as Surrogate Targets

2014 ◽  
Vol 21 (12) ◽  
pp. 1295-1307
Author(s):  
Eva Graczer ◽  
Andras Bacso ◽  
Denes Konya ◽  
Adrian Kazi ◽  
Tibor Soos ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ph Dependence ◽  
Thermus Thermophilus ◽  
Size Exclusion ◽  
Kinetic Properties ◽  
Inhibitory Effects ◽  
Tetrameric Structure ◽  
Exclusion Chromatography ◽  
The Stability ◽  
Isopropylmalate Dehydrogenase

3-Isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (Mtb) may be a target for specific drugs against this pathogenic bacterium. We have expressed and purified Mtb IPMDH and determined its physicalchemical and enzymological properties. Size-exclusion chromatography and dynamic light scattering measurements (DLS) suggest a tetrameric structure for Mtb IPMDH, in contrast to the dimeric structure of most IPMDHs. The kinetic properties (kcat and Kmvalues) of Mtb IPMDH and the pH-dependence of kcat are very similar to both Escherichia coli (Ec) and Thermus thermophilus (Tt) IPMDHs. The stability of Mtb IPMDH in 8 M urea is close to that of the mesophilic counterpart, Ec IPMDH, both of them being much less stable than the thermophilic (Tt) enzyme. Two known IPMDH inhibitors, O-methyl oxalohydroxamate and 3-methylmercaptomalate, have been synthesised. Their inhibitory effects were found to be independent of the origin of IPMDHs. Thus, experiments with either Ec or Tt IPMDH would be equally relevant for designing specific inhibitory drugs against Mtb IPMDH.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii

2021 ◽  
Author(s):  
Quynh DangThu ◽  
Thu-Thuy Nguyen ◽  
Sei-Heon Jang ◽  
ChangWoo Lee
Keyword(s):  
Biochemical Characterization ◽  
Size Exclusion ◽  
Sorbitol Dehydrogenase ◽  
High Catalytic Activity ◽  
Active Site Residues ◽  
Tertiary Structures ◽  
Cold Adapted ◽  
Pseudomonas Mandelii ◽  
Catalytic Active Site ◽  
Exclusion Chromatography

Abstract Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents, and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized D-sorbitol as its carbon source, among the four polyols examined (D-galactitol, D-mannitol, D-sorbitol, or D-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153, and Lys157 as catalytic active site residues and existed as a ∼67 kDa dimer in size-exclusion chromatography. PmSDH converted D-sorbitol to D-fructose using NAD+ as a coenzyme and, vice versa, D-fructose to D-sorbitol using NADH as a coenzyme. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4–25°C. At 40°C, PmSDH was rapidly denatured. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well-suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

scholarly journals Characterization of a monoclonal antibody directed against the biologically active site of human interleukin 1.

1986 ◽  
Vol 163 (2) ◽  
pp. 463-468 ◽  
Author(s):  
A Köck ◽  
M Danner ◽  
B M Stadler ◽  
T A Luger
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Biological Effects ◽  
Interleukin 1 ◽  
Biologically Active ◽  
Size Exclusion ◽  
Fibroblast Proliferation ◽  
Initial Activity ◽  
Exclusion Chromatography

Human IL-1 was successfully used to produce an anti-IL-1 mAb. Anti-IL-1 (IgG2a) blocked IL-1-mediated thymocyte and fibroblast proliferation, but did not interfere with the biological effects of other lymphokines, such as IL-2 or IL-3. The antibody immunoprecipitated biosynthetically radiolabeled 33, 17, and 4 kD IL-1. An immunoadsorbent column yielded 20% of initial activity, and upon HPLC size-exclusion chromatography, affinity-purified IL-1 had a molecular mass of approximately 4 kD. These results provide first evidence of a monoclonal anti-IL-1 that reacts with different species of IL-1 and apparently binds to an epitope close to the active site of IL-1. Thus, anti-IL-1 IgG may be very helpful for further investigations of the molecular as well as biological characteristics of IL-1 and related mediators.

scholarly journals Dimerization of Human Angiogenin and of Variants Involved in Neurodegenerative Diseases

2021 ◽  
Vol 22 (18) ◽  
pp. 10068
Author(s):  
Sabrina Fasoli ◽  
Ilaria Bettin ◽  
Riccardo Montioli ◽  
Andrea Fagagnini ◽  
Daniele Peterle ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Three Dimensional ◽  
Size Exclusion ◽  
Active Site Residues ◽  
Ribonucleolytic Activity ◽  
Pathogenic Variants ◽  
Neuronal Progenitors ◽  
Tumor Tissues ◽  
Exclusion Chromatography ◽  
The Central Nervous System

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies.

Purification and characterization of guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi

Parasitology ◽  
2014 ◽  
Vol 141 (10) ◽  
pp. 1341-1352 ◽  
Author(s):  
SMITA GUPTA ◽  
SUNITA YADAV ◽  
NIDHI SINGH ◽  
ANITA VERMA ◽  
IMRAN SIDDIQI ◽  
...  
Keyword(s):  
Homology Modelling ◽  
Substrate Binding ◽  
Brugia Malayi ◽  
Catalytic Efficiency ◽  
Size Exclusion ◽  
Kinetic Properties ◽  
Guanylate Kinase ◽  
Native Molecular Mass ◽  
Exclusion Chromatography ◽  
Kinase A

SUMMARYGuanylate kinase, a nucleoside monophosphate kinase of Brugia malayi which is involved in reversible transfer of phosphate groups from ATP to GMP, was cloned, expressed and characterized. The native molecular mass of BmGK was found to be 45 kDa as determined by size exclusion chromatography and glutaraldehyde cross-linking which revealed that the protein is homodimer in nature. This is a unique characteristic among known eukaryotic GKs. GMP and ATP served as the most effective phosphate acceptor and donor, respectively. Recombinant BmGK utilized both GMP and dGMP, as substrates showing Km values of 30 and 38 μm, respectively. Free Mg+2 (un-complexed to ATP) and GTP play a regulatory role in catalysis of BmGK. The enzyme showed higher catalytic efficiency as compared with human GK and showed ternary complex (BmGK-GMP-ATP) formation with sequential substrate binding. The secondary structure of BmGK consisted of 45% α-helices, 18% β-sheets as revealed by CD analysis. Homology modelling and docking with GMP revealed conserved substrate binding residues with slight differences. Differences in kinetic properties and oligomerization of BmGK compared with human GK can provide the way for design of parasite-specific inhibitors.

Active Site Residues of Glutamate Racemase†

Biochemistry ◽  
2001 ◽  
Vol 40 (21) ◽  
pp. 6199-6204 ◽  
Author(s):  
Suzana Glavas ◽  
Martin E. Tanner
Keyword(s):  
Active Site ◽  
Active Site Residues ◽  
Glutamate Racemase

scholarly journals Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase.

1997 ◽  
Vol 44 (4) ◽  
pp. 659-672 ◽  
Author(s):  
Z Zhang ◽  
K Ostanin ◽  
R L Van Etten
Keyword(s):  
Acid Phosphatase ◽  
Active Site ◽  
Ph Dependence ◽  
Prostatic Acid Phosphatase ◽  
Competitive Inhibitor ◽  
Covalent Modification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Phosphate Binding

Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.

scholarly journals Modification of catalytically important carboxy residues in endoglucanase D from Clostridium thermocellum

1992 ◽  
Vol 285 (1) ◽  
pp. 319-324 ◽  
Author(s):  
P Tomme ◽  
J van Beeumen ◽  
M Claeyssens
Keyword(s):  
Active Site ◽  
Clostridium Thermocellum ◽  
Ph Dependence ◽  
Reversed Phase ◽  
Spectrophotometric Analysis ◽  
Active Site Residues ◽  
Terminal Sequence ◽  
Local Conservation ◽  
Woodward’S Reagent K ◽  
Phase Liquid

Endoglucanase D (EC 3.2.1.4; EGD) from Clostridium thermocellum is rapidly (k = 216 M-1.min-1) and almost completely (greater than 95%) inactivated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulphonate). Spectrophotometric analysis at 340 nm reveals that eight carboxy residues react, whereas specific ligands protect one residue against modification. The enzyme retains it full activity under the latter conditions. The kinetics and pH-dependence of inactivation point towards the involvement of one or more essential carboxy groups with a pKa of 5.7-5.8. Samples modified in the absence or presence of ligand were analysed by reversed-phase liquid chromatography after proteolysis with subtilisin. Dual-wavelength monitoring at 214 and 340 nm during this fractionation leads to the identification of a putatively active-site peptide (Gly-508-Ala-562) which was further characterized by amino acid and partial N-terminal sequence analyses. Asp-546 and Glu-555 are postulated as possible active-site residues. This follows from alignments using ten endoglucanase sequences belonging to the same family. Strong local conservation suggests that this C-terminal sequence is structurally and/or functionally important.

scholarly journals Organophosphorus acid anhydrolase fromAlteromonas macleodii: structural study and functional relationship to prolidases

2013 ◽  
Vol 69 (4) ◽  
pp. 346-354 ◽  
Author(s):  
Andrea Štěpánková ◽  
Jarmila Dušková ◽  
Tereza Skálová ◽  
Jindřich Hašek ◽  
Tomáš Koval' ◽  
...  
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Cell Parameters ◽  
Bacterial Enzyme ◽  
Organophosphorus Acid Anhydrolase ◽  
Full Structure ◽  
Exclusion Chromatography ◽  
Pita Bread

The bacterial enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The full three-dimensional structure of the manganese-dependent OPAA enzyme is presented for the first time. This enzyme, which was originally isolated from the marine bacteriumAlteromonas macleodii, was prepared recombinantly inEscherichia coli. The crystal structure was determined at 1.8 Å resolution in space groupC2, with unit-cell parametersa= 133.8,b= 49.2,c= 97.3 Å, β = 125.0°. The enzyme forms dimers and their existence in solution was confirmed by dynamic light scattering and size-exclusion chromatography. The enzyme shares the pita-bread fold of its C-terminal domain with related prolidases. The binuclear manganese centre is located in the active site within the pita-bread domain. Moreover, an Ni2+ion from purification was localized according to anomalous signal. This study presents the full structure of this enzyme with complete surroundings of the active site and provides a critical analysis of its relationship to prolidases.

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