Structure, interactions and action of Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase

2018 ◽  
Vol 475 (15) ◽  
pp. 2457-2471 ◽  
Author(s):  
Rajapiramuthu Srikalaivani ◽  
Amrita Singh ◽  
Mamannamana Vijayan ◽  
Avadhesha Surolia
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Mutational Analysis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Native Page ◽  
Acid Dehydrogenase ◽  
Binary Complexes ◽  
Solution Studies ◽  
Hydroxyisobutyric Acid

Biochemical and crystallographic studies on Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase (MtHIBADH), a member of the 3-hydroxyacid dehydrogenase superfamily, have been carried out. Gel filtration and blue native PAGE of MtHIBADH show that the enzyme is a dimer. The enzyme preferentially uses NAD+ as the cofactor and is specific to S-hydroxyisobutyric acid (HIBA). It can also use R-HIBA, l-serine and 3-hydroxypropanoic acid (3-HP) as substrates, but with much less efficiency. The pH optimum for activity is ∼11. Structures of the native enzyme, the holoenzyme, binary complexes with NAD+, S-HIBA, R-HIBA, l-serine and 3-HP and ternary complexes involving the substrates and NAD+ have been determined. None of the already known structures of HIBADH contain a substrate molecule at the binding site. The structures reported here provide for the first time, among other things, a clear indication of the location and interactions of the substrates at the active site. They also define the entrance of the substrates to the active site region. The structures provide information on the role of specific residues at the active site and the entrance. The results obtained from crystal structures are consistent with solution studies including mutational analysis. They lead to the proposal of a plausible mechanism of the action of the enzyme.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Investigation of the active site of oligosaccharyltransferase from pig liver using synthetic tripeptides as tools

1995 ◽  
Vol 312 (3) ◽  
pp. 979-985 ◽  
Author(s):  
E Bause ◽  
W Breuer ◽  
S Peters
Keyword(s):  
Hydroxy Group ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Endoplasmic Reticulum Membrane ◽  
Hydrogen Binding ◽  
Ph Optimum ◽  
Pig Liver ◽  
Acceptor Activity ◽  
Amide Carbonyl ◽  
Amide Carbonyl Group

Oligosaccharyltransferase (OST), an integral component of the endoplasmic-reticulum membrane, catalyses the transfer of dolichyl diphosphate-linked oligosaccharides to specific asparagine residues forming part of the Asn-Xaa-Thr/Ser sequence. We have studied the binding and catalytic properties of the enzyme from pig liver using peptide analogues derived from the acceptor peptide N-benzoyl-Asn-Gly-Thr-NHCH3 by replacing either asparagine or threonine with amino acids differing in size, stereochemistry, polarity and ionic properties. Acceptor studies showed that analogues of asparagine and threonine with bulkier side chains impaired recognition by OST. Reduction of the beta-amide carbonyl group of asparagine yielded a derivative that, although not glycosylated, was strongly inhibitory (50% inhibition at approximately 140 microM). This inhibition may be due to ion-pair formation involving the NH3+ group and a negatively charged base at the active site. Hydroxylation of asparagine at the beta-C position increased Km and decreased Vmax, indicating an effect on both binding and catalysis. The threo configuration at the beta-C atom of the hydroxyamino acid was essential for substrate binding. A peptide derivative obtained by replacement of the threonine beta-hydroxy group with an NH2 group was found to display acceptor activity. This shows that the primary amine is able to mimic the hydroxy group during transglycosylation. The pH optimum with this derivative is shifted by approximately 1 pH unit towards the basic region, indicating that the neutral NH2 group is the reactive species. The various data are discussed in terms of the catalytic mechanism of OST, particular emphasis being placed on the role of threonine/serine in increasing the nucleophilicity of the beta-amide of asparagine through hydrogen-binding.

scholarly journals Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis

1994 ◽  
Vol 297 (2) ◽  
pp. 351-357 ◽  
Author(s):  
A Lemassu ◽  
M Daffé
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mimicry ◽  
Degradation Products ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Structural Features ◽  
Two Dimensional ◽  
Phagocytic Cells ◽  
Molecular Masses ◽  
Branched Structure

The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.

embB Gene association with Ethambutol drug Resistance in Mycobacterium Tuberculosis Patients

2021 ◽  
Vol 15 (12) ◽  
pp. 3273-3276
Author(s):  
Sana Hafeez ◽  
Haleema Sajid ◽  
Farouk Qamar Malik ◽  
Imran Ali Zaidi ◽  
Sobia Niaz ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Codon Position ◽  
Mutational Analysis ◽  
Transmission Rate ◽  
Drug Resistant ◽  
Tuberculosis Patients ◽  
Two Samples ◽  
Suspected Tuberculosis ◽  
Embb Gene

Background: Tuberculosis (TB) is fatal and life threatening infectious disease. The transmission rate of tuberculosis is very high. Various drugs are used as treatment for TB. Recently it has been observed that one of the most important factor for fast TB spread is development of anti-TB drug resistant mycobacterium tuberculosis (MTB). Various combination of drugs like isoniazid (INH), rifampicin (RIF), Streptomycin(SM), pyrazinamide (PZA) or ethambutol (EMB) are in global use for TB treatment. Improper usage of these drugs makes the person prone to develop anti-TB drug resistant tuberculosis. Aim: To evaluate association of embB gene with ethambutol resistance in Mycobacterium Tuberculosis. Methods: 104 Specimens of sputum from suspected tuberculosis patients were processed for inoculation in Lowenstein J Medium after it has been decontaminated properly. Kit method by using QIAamp DNA Mini kit was utilized for extraction of DNA. Then region from base 6953 to 10249 of embB gene was amplified through PCR and then followed by sequencing with the aid of softwares blast2seq and ClustalW2. Three primer sets were utilized to amplify embB gene. Ethambutol (EMB) Resistant MTB specimens were processed to study mutation in embB gene. Results: Out of the total 104 sputum specimens, 14 samples were found to have ethambutol resistance. These 14 samples were then processed for mutational analysis. DNA sequence analysis of these 14 samples confirmed embB gene mutation in 10 samples. Mutational analysis revealed that 08 samples showed mutation at codon 306 and two samples showed mutation at 319 codon. The reported mutation Methionine →Isoleucine was seen in 07 samples with ATG codon replaced by ATA codon at codon position 306. One sample showed mutation as Methionine →Isoleucine with ATG codon replaced by ATC codon at codon position 306. Two samples showed mutation as Tyrosine →Serine with TAT codon replaced by TCT at 319 codon position in embB gene. Conclusion: This study concludes that mutation of certain genes particularly point mutation of embB gene at codon 306 and 319 is associated with drug resistance of ethambutol in ethambutol resistant mycobacterium tuberculosis patients. Keywords: Ethambutol, embB gene, Mycobacterium tuberculosis.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

1984 ◽  
Vol 4 (6) ◽  
pp. 1003-1012
Author(s):  
R L Nelson ◽  
P E Branton
Keyword(s):  
Chicken Embryo ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Soluble Form ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Phosphoprotein Phosphatases ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

2014 ◽  
Vol 70 (12) ◽  
pp. 3212-3225 ◽  
Author(s):  
Tiila-Riikka Kiema ◽  
Rajesh K. Harijan ◽  
Malgorzata Strozyk ◽  
Toshiyuki Fukao ◽  
Stefan E. H. Alexson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reaction Mechanism ◽  
Active Site ◽  
Quaternary Structure ◽  
Fatty Acyl ◽  
Physiological Importance ◽  
Loop Domain ◽  
Solution Studies ◽  
Hydrolysis Of ◽  
Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

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