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.

scholarly journals Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans

2005 ◽  
Vol 71 (9) ◽  
pp. 5501-5510 ◽  
Author(s):  
Sandra W. A. Hinz ◽  
Marieke I. Pastink ◽  
Lambertus A. M. van den Broek ◽  
Jean-Paul Vincken ◽  
Alphons G. J. Voragen
Keyword(s):  
Molecular Mass ◽  
Transmembrane Domain ◽  
Bifidobacterium Longum ◽  
Cell Extract ◽  
Size Exclusion ◽  
Glycoside Hydrolase Family ◽  
Type I ◽  
Native Molecular Mass ◽  
Exclusion Chromatography ◽  
Hydrolase Family

ABSTRACT A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(−) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and V max for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (β1→4)galactans and (β1→4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (β1→3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

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 Functional and molecular modelling studies of two hereditary fructose intolerance-causing mutations at arginine 303 in human liver aldolase

2000 ◽  
Vol 350 (3) ◽  
pp. 823-828 ◽  
Author(s):  
Rita SANTAMARIA ◽  
Gabriella ESPOSITO ◽  
Luigi VITAGLIANO ◽  
Vincenza RACE ◽  
Immacolata PAGLIONICO ◽  
...  
Keyword(s):  
Homology Modelling ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Kinetic Properties ◽  
Wild Type ◽  
Hereditary Fructose Intolerance ◽  
Fructose Intolerance ◽  
Aldolase B ◽  
Fructose 1,6 Bisphosphate

We have identified a novel hereditary fructose intolerance mutation in the aldolase B gene (i.e. liver aldolase) that causes an arginine-to-glutamine substitution at residue 303 (Arg303 → Gln). We previously described another mutation (Arg303 → Trp) at the same residue. We have expressed the wild-type protein and the two mutated proteins and characterized their kinetic properties. The catalytic efficiency of protein Gln303 is approx. 1/100 that of the wild-type for substrates fructose 1,6-bisphosphate and fructose 1-phosphate. The Trp303 enzyme has a catalytic efficiency approx. 1/4800 that of the wild-type for fructose 1,6-bisphosphate; no activity was detected with fructose 1-phosphate. The mutation Arg303 → Trp thus substitution impairs enzyme activity more than Arg303 → Gln. Three-dimensional models of wild-type, Trp303 and Gln303 aldolase B generated by homology-modelling techniques suggest that, because of its larger size, tryptophan exerts a greater deranging effect than glutamine on the enzyme's three-dimensional structure. Our results show that the Arg303 → Gln substitution is a novel mutation causing hereditary fructose intolerance and provide a functional demonstration that Arg303, a conserved residue in all vertebrate aldolases, has a dominant role in substrate binding during enzyme catalysis.

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.

Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DP8)

2010 ◽  
Vol 391 (8) ◽  
Author(s):  
Melissa R. Pitman ◽  
R. Ian Menz ◽  
Catherine A. Abbott
Keyword(s):  
Biological Function ◽  
Homology Modelling ◽  
Dipeptidyl Peptidase ◽  
Fibroblast Activation Protein ◽  
Size Exclusion ◽  
Dimer Interface ◽  
Endopeptidase Activity ◽  
Exclusion Chromatography ◽  
Hydrophilic Residues ◽  
Substrate Kinetics

Abstract Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8D772, DP8Y315, DP8H434 and DP8D435 and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8D772A/E/S/V affected catalysis but did not confer endopeptidase activity. Mutations of DP8H434F, DP8D435F and DP8Y315F reduced catalytic activity. Furthermore, mutations to DP8D772A/E/S/V, DP8H434F, DP8D435F and DP8Y315F affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8D772, DP8H434 and DP8D435 were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.

Phenolic Resins – Characterizations and Kinetic Studies of Different Resols Prepared with Different Catalysts and Formaldehyde/Phenol Ratios (I)

2002 ◽  
Vol 10 (5) ◽  
pp. 341-360 ◽  
Author(s):  
J. Bouajila ◽  
G. Raffin ◽  
H. Waton ◽  
C. Sanglar ◽  
J.O. Païsse ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Size Exclusion ◽  
Kinetic Properties ◽  
Reaction Products ◽  
Phenolic Resins ◽  
Chemical Assay ◽  
Exclusion Chromatography ◽  
Kinetics Of Formation ◽  
Minor Compounds

The physicochemical and kinetic properties of resols prepared with different catalysts (NaOH, LiOH and Ba(OH)2) and variable formaldehyde/phenol ratios (2.5 £ R £ 3.5) were followed to determine their effects on the mechanisms and reaction products at a fixed pH and temperature. Kinetic monitoring and quantification of residual monomers were carried out by liquid chromatography coupled with mass spectrometry (LC/UV/MS), by 13C nuclear magnetic resonance (NMR) and by chemical assay for formaldehyde. Oligomer formation (n ≥ 2) was determined by LC/UV/MS, size exclusion chromatography (SEC) and 13C NMR. It was found that minor compounds form during syntheses (phenol methanol hemiacetals, hemiacetals of phenol and of oligomers…) and that the ratio R affects primarily the kinetics of formation of monomers and oligomers, in contrast to the catalysts that modify reaction mechanisms. The understanding of the structure of the resols was an important step for the determination of the final properties of the material.

scholarly journals Structural and biochemical analysis of ATPase activity and EsxAB substrate binding of M. tuberculosis EccCb1 enzyme

2021 ◽  
Author(s):  
Arkita Bandyopadyay ◽  
Ajay Kumar Saxena
Keyword(s):  
Atpase Activity ◽  
Biochemical Analysis ◽  
Substrate Binding ◽  
Catalytic Efficiency ◽  
Size Exclusion ◽  
Enzyme Dynamics ◽  
Low Resolution ◽  
Promising Target ◽  
Binding Pockets ◽  
Resolution Structure

The EccC enzyme of M. tuberculosis ESX-1 system is a promising target for antivirulence drug development. The EccC enzyme comprises two polypeptides (i) EccCa1, a membrane bound enzyme having two ATPase domains D2 & D3 (ii) cytosolic EccCb, which contains two ATPase domains. In current study, we have analyzed the low-resolution structure of EccCb1, performed ATPase activity and EsxAB substrate binding analysis. The EccCb1 enzyme eluted as oligomer from size exclusion column and small angle X-ray scattering analysis revealed the double hexameric structure in solution. The EccCb1 enzyme showed catalytic efficiency (kcat/KM)~ 0.020 micromolar-1 min-1, however ~3.7 fold lower than its D2 and ~1.7 fold lower than D3 domains respectively. The D2 and D3 domains exhibited the ATPase activity and mutation of residues involved in ATP+Mg2+ binding have yielded 56-94% reduction in catalytic efficiency for both D2 and D3 domains. The EccCb1 binds the EsxAB substrate with KD ~ 11.4 nM via specific groove located at C-terminal region of D3 domain. ATP binding to EccCb1 enhanced the EsxAB substrate binding by ~ 3 fold, indicating ATPase energy involvement in EsxAB substrate translocation. We modeled the dodecameric EccCb1+EsxAB+ ATP+Mg2+ complex, which showed the binding pockets involved in ATP+Mg2+ and EsxAB substrate binding. The enzyme dynamics involved in ATP+Mg2+ and EsxAB substrate recognition were identified and showed the enhanced stability of EccCb1 enzyme as a result of ligand binding. Overall, our structural and biochemical analysis showed the low-resolution structure and mechanism involved in ATPase activity and EsxAB substrate binding and dynamics involved in EsxAB substrate and ATP+Mg2+ recognition. Overall, our structural and biochemical data on EccCb1 will contribute significantly in development of antivirulence inhibitors, which will prevent virulence factor secretion by M. tuberculosis ESX-1 system.

scholarly journals The structure of procaspase 6 is similar to that of active mature caspase 6

2002 ◽  
Vol 364 (3) ◽  
pp. 629-634 ◽  
Author(s):  
Byoung Heon KANG ◽  
Eunsil KO ◽  
Oh-Keun KWON ◽  
Kwan Yong CHOI
Keyword(s):  
Binding Site ◽  
Structural Characteristics ◽  
Substrate Binding ◽  
Size Exclusion ◽  
Activation Mechanism ◽  
Tryptophan Residue ◽  
Substrate Binding Site ◽  
Genes Encoding ◽  
Exclusion Chromatography ◽  
Caspase 6

To investigate the structural characteristics and activation mechanism of the precursor caspase, genes encoding the inactive pro-form and the active mature form of caspase 6 were expressed in Escherichia coli and the proteins of both forms were purified to homogeneity. The structure of each protein was characterized by chemical cross-linking, size-exclusion chromatography, CD and fluorescence spectroscopies. The pro-form caspase 6 exhibits a dimeric structure and its overall secondary structure was found to be similar to that of the mature caspase 6. Upon the maturation of procaspase 6, the maximum fluorescence wavelength λmax was red-shifted from 330 to 337nm and the fluorescence intensity of λmax was increased. This fluorescence spectral change indicates that the environment of a tryptophan residue in the substrate-binding site can be changed to a more polar one when the procaspase 6 is processed. Taken together, our results strongly demonstrate that precursor caspase 6 exists as a dimer and its overall structure is similar to that of the active caspase 6. Our results also suggest that the local conformational change at the substrate-binding site, with no drastic change in the overall structure, seems to enable precursor caspase 6 to become the active mature enzyme.

scholarly journals The DnaK chaperones from the archaeon Methanosarcina mazei and the bacterium Escherichia coli have different substrate specificities.

2007 ◽  
Vol 54 (3) ◽  
pp. 509-522 ◽  
Author(s):  
Michal A Zmijewski ◽  
Joanna Skórko-Glonek ◽  
Fabio Tanfani ◽  
Bogdan Banecki ◽  
Agnieszka Kotlarz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Binding ◽  
Active Form ◽  
Size Exclusion ◽  
Methanosarcina Mazei ◽  
Binding Domains ◽  
Substrate Peptide ◽  
Exclusion Chromatography

Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK(Mm)) and the DnaK from the bacterium Escherichia coli (DnaK(Ec)) were functionally similar when assayed in vitro but DnaK(Mm) failed to substitute for DnaK(Ec) in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK(Mm) and DnaK(Ec). DnaK(Mm) showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK(Ec). Furthermore, it was unable to negatively regulate the E. coli sigma32 transcription factor level under heat shock conditions and poorly bound purified sigma32, which is a native substrate of DnaK(Ec). These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK(Mm) showed some structural differences in the substrate-binding domains of DnaK(Mm) and DnaK(Ec), which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK(Mm) was found preferably in high molecular mass oligomeric forms, contrary to DnaK(Ec). Oligomers of DnaK(Mm) could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK(Mm).

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