Characterization of a novel exo-N-acetyl-β-d-glucosaminidase from the thermotolerant Bacillus sp. NCIM 5120

Amutha, B.; Khire, J. M.; Khan, M. Islam

doi:10.1016/s0304-4165(98)00081-6

Cited by 13 publications

(6 citation statements)

References 36 publications

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“…Such biphasicity in the energy of activation has not previously been reported for any RecA‐like protein. However, discontinuous Arrhenius plots have been observed for some membrane proteins [53] and for certain enzymes from thermophiles [54] and hibernating mammals [55]. A possible hypothesis for the break in the Arrhenius plot is the existence of two conformational states of the enzyme above and below the break point, each with a different catalytic competence [56].…”

Section: Discussionmentioning

confidence: 99%

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA‐dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 °C

Spies

Kil

Masui

et al. 2000

European Journal of Biochemistry

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The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 8C. It also showed high stability of 18.3 kcal´mol 21(76.5 kJ´mol 21) at 25 8C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecAlike proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 8C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 8C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 8C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 8C being the critical threshold temperature.Keywords: Arrhenius plot; DNA-dependent ATPase; homologous genetic recombination; Pyrobaculum islandicum; RadA.Homologous or general recombination is a fundamental mechanism and a vital process in most living organisms. The RecA-like recombinational proteins play a central role in the process of genetic recombination by bringing two interacting DNA molecules together, searching for homology and exchanging the DNA strands [1,2]. Upon binding ATP, RecA protein in bacteria and Rad51 protein in eukaryotes organize a highly structured nucleoprotein filament which makes the DNA available for homologous pairing [1,3±7]. Recently, the RecA paradigm of homologous recombination was extended to all three domains of life, Bacteria, Eukarya and Archaea [8±11]. The mechanisms of homologous recombination and DNA repair have been extensively described for prokaryotes [1±5] and eukaryotes [7,12,13], but little is known about these mechanisms in archaea. Many RecA homologs have been isolated from prokaryotes and eukaryotes, but only recently have sequences encoding proteins similar to RecA and Rad51 been found in the third phylogenetically divergent group, Archaea [8,11,14±17]. Two types of sequence encoding putative RecA-like proteins have been discovered in different archaeal species: polypeptides of 320±350 amino acids (RadA proteins) [8,11], and those of 210±230 amino acids (RadB proteins) [14]. In addition, both types of sequence have been found in archaeal strains, complete genomes of which are now available [15±17]. The reported putative proteins encoded by radA genes are more similar in sequence to Rad51 and Dmc1 proteins than to RecA proteins [8,9,11]. Within the minimal core region, in whic...

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Section: Discussionmentioning

confidence: 99%

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA‐dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 °C

Spies

Kil

Masui

et al. 2000

European Journal of Biochemistry

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“…It was however insensitive to EDTA suggesting it is not a metalloenzyme. This property though, is not uncommon to most studied NAGases [8][9][10].…”

Section: Discussionmentioning

confidence: 85%

“…The enzyme has been described in bacteria, fungi, T. cruzi, and Trichomonas [8][9][10][11]. We report here a novel NAGase from B. arietans and possible toxinomous properties.…”

Section: Discussionmentioning

confidence: 88%

“…The enzyme from T. cruzi with a pH optimum of 7.5 has a K M of 1.25 mM for MUGlcNAc. The K M for Bacillus NAGase is 0.34 mM [8][9][10][11].…”

Section: Discussionmentioning

confidence: 99%

“…Exo-N-acetyl-␤-D-glucosaminidase (␤-1,2-acetamido-2-deoxy-D-glucoside acetamido deoxyglucohydrolase EC 3.2.1.30) catalyses the hydrolysis of terminally occurring (␤-1-4) linked N-acetylglucosaminyl residues from nonreducing ends of glycoconjugates [8]. Their functions vary from playing an important role during cell wall development in bacteria [9], autolysing activity during fungal cell development, to molting in insects [10].…”

Section: Introductionmentioning

confidence: 99%

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N‐Acetyl α‐D‐Glucosaminidase from the Venom of African Puff Adder (Bitis Arietans)

Nok

Shuaibu

Choudhry

et al. 2001

J Biochem & Molecular Tox

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The activity of N-acetyl-alpha-D-glucosaminidase from venom of the African puff adder (Bitis arietans) has been detected. The enzyme from the venom was purified by chromatography on Q-sepharose, CM-cellulose, and N-acetyl-alpha-D-glucosamine-agarose affinity column. The enzyme has a molecular weight of 102 kDa determined by size exclusion chromatography on Sephacryl 200. It migrated as a 51-kDa band on SDS polyacrylamide gels. The enzyme is maximally active at pH 5.5 and 40 degrees C. The B. arietans NAGase hydrolyzed exclusively terminally linked alpha-(1-4) GlcNAc residues from nonreducing ends of oligosaccharides. It hydrolysed chito-oligosaccharide, MU-GlcNAc and chitobiose with K(M) values of 0.15 mM and 1.22 mM, respectively. Swollen chitin and oligosaccharide above (GlcNAc)(4) were not hydrolysed by the enzyme. B. arietans NAGase was strongly inhibited noncompetitively by Hg(2+), competitively by 1-thio-beta-D-GlcNAc and N-acetyl glucosamine (NAG) with K(i) of 0.55, 0.25 and 8 mM, respectively. Colombin the active component of antivenom preparation from Aristolodia albida inhibited the enzyme competitively with K(i) of 0.6 mM. Delineation of the active site by chemical modification revealed the involvement of His and Trp in the catalysis of the enzyme.

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[NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics

et al. 2003

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Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

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Characterization of a novel exo-N-acetyl-β-d-glucosaminidase from the thermotolerant Bacillus sp. NCIM 5120

Cited by 13 publications

References 36 publications

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA‐dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 °C

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA‐dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 °C

N‐Acetyl α‐D‐Glucosaminidase from the Venom of African Puff Adder (Bitis Arietans)

[NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics

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