scholarly journals Purification and characterization of methionine γ-lyase from Trichomonas vaginalis

1991 ◽  
Vol 279 (3) ◽  
pp. 675-682 ◽  
Author(s):  
B C Lockwood ◽  
G H Coombs
Keyword(s):  
Molecular Mass ◽  
Trichomonas Vaginalis ◽  
Protozoan Parasite ◽  
Purification And Characterization ◽  
Native Molecular Mass ◽  
Substrate Analogues ◽  
Beta Elimination ◽  
Derivatives Of ◽  
Methionine Gamma Lyase

Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from the anaerobic protozoan parasite Trichomonas vaginalis by a series of f.p.l.c. procedures. The enzyme catalyses alpha gamma- and alpha beta-elimination reactions of a number of derivatives of methionine and cysteine. It also catalyses gamma-replacement reactions of the thiomethyl group of methionine, homocysteine and ethionine to yield the corresponding S-substituted homocysteine derivative. The enzyme is pyridoxal 5′-phosphate-dependent, has a native molecular mass of approx. 160 kDa and consists of four apparently identical subunits of molecular mass 43-45 kDa. The absorption spectrum of the enzyme is typical of those obtained for other pyridoxal 5′-phosphate-dependent enzymes, and the holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of the cofactor. The enzyme activity is significantly affected by carbonyl and thiol reagents, is competitively inhibited by a number of substrate analogues and is completely inactivated by the suicide inhibitor DL-propargylglycine. The T. vaginalis enzyme is similar, in terms of activity and properties, to the enzymes found in a number of species of bacteria that metabolize methionine under anaerobic conditions. It is suggested that methionine catabolism may be of particular importance to the survival of T. vaginalis under microaerophilic conditions in its host.

scholarly journals Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3083-3091 ◽  
Author(s):  
Jaigeeth Deveryshetty ◽  
Prashant S. Phale
Keyword(s):  
Molecular Mass ◽  
Substrate Inhibition ◽  
Enzyme Reaction ◽  
Sole Source ◽  
Purification And Characterization ◽  
Spectral Changes ◽  
Native Molecular Mass ◽  
Naphthoic Acid ◽  
Key Enzyme

Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy via the ‘phthalic acid’ route. The key enzyme, 1-hydroxy-2-naphthoic acid dioxygenase (1-HNDO, EC 1.13.11.38), was purified to homogeneity using a 3-hydroxy-2-naphthoic acid (3-H2NA)-affinity matrix. The enzyme was a homotetramer with a native molecular mass of 160 kDa and subunit molecular mass of ∼39 kDa. It required Fe(II) as the cofactor and was specific for 1-hydroxy-2-naphthoic acid (1-H2NA), with K m 13.5 μM and V max 114 μmol min−1 mg−1. 1-HNDO failed to show activity with gentisic acid, salicylic acid and other hydroxynaphthoic acids tested. Interestingly, the enzyme showed substrate inhibition with a K i of 116 μM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a K i of 24 μM. Based on the pH-dependent spectral changes, the enzyme reaction product was identified as 2-carboxybenzalpyruvic acid. Under anaerobic conditions, the enzyme failed to convert 1-H2NA to 2-carboxybenzalpyruvic acid. Stoichiometric studies showed the incorporation of 1 mol O2 into the substrate to yield 1 mol product. These results suggest that 1-HNDO from Pseudomonas sp. strain PPD is an extradiol-type ring-cleaving dioxygenase.

Purification and characterization of a lectin from Xanthomonas campestris NCIM 5028

1996 ◽  
Vol 42 (6) ◽  
pp. 609-612 ◽  
Author(s):  
Bhagyashree Joshi ◽  
Jayant M. Khire ◽  
Hephzibah SivaRaman ◽  
M. Islam Khan
Keyword(s):  
Molecular Mass ◽  
Host Plant ◽  
Xanthomonas Campestris ◽  
Simple Procedure ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Molecular Masses ◽  
Lectin Purification

A lectin was isolated from culture filtrates of Xanthomonas campestris NCIM 5028, by a simple procedure of hydrophobic chromatography on phenyl-Sepharose after ammonium sulphate precipitation. The lectin was a heterodimer, with subunit molecular masses of 30 000 and 28 000. Gel filtration on S-300 column, calibrated with markers, showed its molecular mass to be approximately 70 000. Its isoelectric point was 7.2. The agglutination of the rabbit erythrocytes by the lectin was inhibited by fetuin glycopeptides and host plant (Brassica oleracea) extracts.Key words: Xanthomonas campestris, lectin, purification.

Purification and Characterization of Gentisate 1,2-Dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869

1999 ◽  
Vol 65 (3) ◽  
pp. 946-950 ◽  
Author(s):  
Yongmei Feng ◽  
Hoon Eng Khoo ◽  
Chit Laa Poh
Keyword(s):  
Pseudomonas Putida ◽  
Molecular Mass ◽  
Amino Acid Residues ◽  
Stability Range ◽  
Native Molecular Mass ◽  
Heat Stable ◽  
A Subunit ◽  
Pseudomonas Alcaligenes ◽  
Similar Kinetic

ABSTRACT Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1,2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1,2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Km s of 92 and 143 μM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with k cat/Km values of 44.08 × 104 s−1 M−1 for the P25X enzyme and 39.34 × 104 s−1M−1 for the P35X enzyme. Higherk cat/Km values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1,2-dioxygenases was around 50°C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+. Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.

Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11

2006 ◽  
Vol 52 (2) ◽  
pp. 157-168 ◽  
Author(s):  
Subhas Das ◽  
Dileep Kumar Singh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mass ◽  
Half Life ◽  
Indoleacetic Acid ◽  
Bacterial Strains ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Microbial Biodegradation ◽  
Membrane Bound

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34–41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%–16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life = 7.3 h) than that from SBL11 (half-life = 6.4 h at 50 °C), while both showed the same temperature optimum of 37 °C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.Key words: biodegradation, monocrotophos, phosphotriesterase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11.

scholarly journals Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides

1994 ◽  
Vol 300 (2) ◽  
pp. 557-565 ◽  
Author(s):  
C Alban ◽  
P Baldet ◽  
R Douce
Keyword(s):  
Molecular Mass ◽  
Subunit Structure ◽  
Acetyl Coa Carboxylase ◽  
Leaf Extracts ◽  
Acetyl Coa ◽  
Native Molecular Mass ◽  
Molecular Masses ◽  
Minor Form ◽  
A Minor

Young pea leaves contain two structurally different forms of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase). A minor form, which accounted for about 20% of the total ACCase activity in the whole leaf, was detected in the epidermal tissue. This enzyme was soluble and was purified to homogeneity from young pea leaf extracts. It consisted of a dimer of two identical biotinyl subunits of molecular mass 220 kDa. In this respect, this multifunctional enzyme was comparable with that described in other plants and in other eukaryotes. A predominant form was present in both the epidermal and mesophyll tissues. In mesophyll protoplasts, ACCase was detected exclusively in the soluble phase of chloroplasts. This enzyme was partially purified from pea chloroplasts and consisted of a freely dissociating complex, the activity of which may be restored by combination of its separated constituents. The partially purified enzyme was composed of several subunits of molecular masses ranging from 32 to 79 kDa, for a native molecular mass > 600 kDa. One of these subunits, of molecular mass 38 kDa, was biotinylated. This complex subunit structure was comparable with that of microorganisms and was referred to as a ‘prokaryotic’ form of ACCase. Biochemical parameters were determined for both ACCase forms. Finally, both pea leaf ACCases exhibited different sensitivities towards the grass ACCase herbicide, diclofop. This compound had no effect on the ‘prokaryotic’ form of ACCase, while the ‘eukaryotic’ form was strongly inhibited.

scholarly journals Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

2000 ◽  
Vol 350 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Sebabrata MAHAPATRA ◽  
Sanjib BHAKTA ◽  
Jasimuddin AHAMED ◽  
Joyoti BASU
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Mycobacterium Leprae ◽  
Binding Activity ◽  
High Molecular Mass ◽  
Class A ◽  
C Terminus ◽  
Fusion Junction ◽  
Derivatives Of

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627–4630]. PBP1-Xaa–β-lactamase fusions generated periplasmic β-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [∆M1–R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [∆M1–R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.

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