scholarly journals Characterization of a C—C Bond Hydrolase from Sphingomonas wittichii RW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites

2007 ◽  
Vol 189 (11) ◽  
pp. 4038-4045 ◽  
Author(s):  
Stephen Y. K. Seah ◽  
Jiyuan Ke ◽  
Geoffroy Denis ◽  
Geoff P. Horsman ◽  
Pascal D. Fortin ◽  
...  
Keyword(s):  
Polychlorinated Biphenyl ◽  
Phylogenetic Analyses ◽  
Inverse Pcr ◽  
Gene Encoding ◽  
Hydrophobic Residues ◽  
Burkholderia Xenovorans ◽  
Lower Specificity ◽  
Sphingomonas Wittichii ◽  
Hydrolysis Of

ABSTRACT Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k cat/Km = 1.2 × 107 M−1 s−1) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k cat/Km = 1.7 × 106 M−1 s−1), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

scholarly journals A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2, the Gene Encoding a Fungal Aminodeoxychorismate Lyase

2007 ◽  
Vol 6 (11) ◽  
pp. 2102-2111 ◽  
Author(s):  
Javier Botet ◽  
Laura Mateos ◽  
José L. Revuelta ◽  
María A. Santos
Keyword(s):  
Large Scale ◽  
Phylogenetic Analyses ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Cellular Processes ◽  
Founder Member ◽  
Yeast Genes ◽  
Vacuole Biogenesis ◽  
Reading Frames

ABSTRACT Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.

scholarly journals A novel decrystallizing protein CxEXL22 from Arthrobotrys sp. CX1 capable of synergistically hydrolyzing cellulose with cellulases

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Rong Li ◽  
Yunze Sun ◽  
Yihao Zhou ◽  
Jiawei Gai ◽  
Linlu You ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Hydrophobic Surface ◽  
Crystalline Cellulose ◽  
Crystalline Region ◽  
Strong Activity ◽  
Hydrophobic Residues ◽  
Cellulose Accessibility ◽  
Hydrolysis Of ◽  
Β Sheet

AbstractA novel expansin-like protein (CxEXL22) has been identified and characterized from newly isolated Arthrobotrys sp. CX1 that can cause cellulose decrystallization. Unlike previously reported expansin-like proteins from microbes, CxEXL22 has a parallel β-sheet domain at the N terminal, containing many hydrophobic residues to form the hydrophobic surface as part of the groove. The direct phylogenetic relationship implied the genetic transfers occurred from nematode to nematicidal fungal Arthrobotrys sp. CX1. CxEXL22 showed strong activity for the hydrolysis of hydrogen bonds between cellulose molecules, especially when highly crystalline cellulose was used as substrate. The hydrolysis efficiency of Avicel was increased 7.9-fold after pretreating with CxEXL22. The rupture characterization of crystalline region indicated that CxEXL22 strongly binds cellulose and breaks up hydrogen bonds in the crystalline regions of cellulose to split cellulose chains, causing significant depolymerization to expose much more microfibrils and enhances cellulose accessibility.

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2931-2939 ◽  
Author(s):  
Alex K. Jaques ◽  
Tamo Fukamizo ◽  
Diana Hall ◽  
Richard C. Barton ◽  
Gemma M. Escott ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Wild Type Strain ◽  
Chitinase Activity ◽  
Gene Replacement ◽  
Wild Type ◽  
Gene Encoding ◽  
Batch Cultures ◽  
Putative Active Site ◽  
Hydrolysis Of

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the β-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc4, GlcNAc5 and GlcNAc6 indicated that the predominant reaction involved hydrolysis of GlcNAc2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

scholarly journals Characterization of the Family I inorganic pyrophosphatase fromPyrococcus horikoshiiOT3

Archaea ◽  
2005 ◽  
Vol 1 (6) ◽  
pp. 385-389 ◽  
Author(s):  
Sung-Jong Jeon ◽  
Kazuhiko Ishikawa
Keyword(s):  
Maximum Velocity ◽  
Heat Stability ◽  
Hydrolytic Activity ◽  
Heat Inactivation ◽  
Inorganic Pyrophosphatase ◽  
Gene Encoding ◽  
Sds Page ◽  
The Family ◽  
Hydrolysis Of

A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeonPyrococcus horikoshiiOT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) fromP. horikoshiishowed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase fromP. horikoshii(PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability ofPhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+being most effective; Zn2+, Co2+and Mn2+efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Kmfor pyrophosphate and Mg2+were 113 and 303 µM, respectively; and maximum velocity,Vmax, was estimated at 930 U mg–1.

scholarly journals Acetate Activation inMethanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase

Archaea ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Stefanie Berger ◽  
Cornelia Welte ◽  
Uwe Deppenmeier
Keyword(s):  
Energy Balance ◽  
Thermodynamic Limit ◽  
Major Part ◽  
Acetyl Coa ◽  
Gene Encoding ◽  
Activation Reaction ◽  
Key Enzymes ◽  
Hydrolysis Of ◽  
Acetate Activation

The thermophilic methanogenMethanosaeta thermophilauses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction inMethanosarcina sp.is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction inMt. thermophilaseems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate(PPi)and was only moderately inhibited byPPi. The breakdown ofPPiwas performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part ofPPi(KM=0.27±0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides orPPi. However, it cannot be excluded that otherPPi-dependent enzymes take advantage of the remainingPPiand contribute to the energy balance of the cell.

Phylogeny Characterization of Seb Gene Encoding Enterotoxins in Staphylococcus aureus Isolated from Raw Milk and Cheese

2019 ◽  
Vol 9 (o3) ◽  
Author(s):  
Fatima N. Aziz ◽  
Laith Abdul Hassan Mohammed-Jawad
Keyword(s):  
Staphylococcus Aureus ◽  
Food Poisoning ◽  
Raw Milk ◽  
Staphylococcal Enterotoxin B ◽  
Human Pathogen ◽  
Conventional Pcr ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Gene Encoding

Food poisoning due to the bacteria is a big global problem in economically and human's health. This problem refers to an illness which is due to infection or the toxin exists in nature and the food that use. Milk is considered a nutritious food because it contains proteins and vitamins. The aim of this study is to detect and phylogeny characterization of staphylococcal enterotoxin B gene (Seb). A total of 200 milk and cheese samples were screened. One hundred ten isolates of Staphylococcus aureus pre-confirmed using selective and differential media with biochemical tests. Genomic DNA was extracted from the isolates and the SEB gene detects using conventional PCR with specific primers. Three staphylococcus aureus isolates were found to be positive for Seb gene using PCR and confirmed by sequencing. Sequence homology showed variety range of identity starting from (100% to 38%). Phylogenetic tree analyses show that samples (6 and 5) are correlated with S. epidermidis. This study discovered that isolates (A6-RLQ and A5-RLQ) are significantly clustered in a group with non- human pathogen Staphylococcus agnetis.

scholarly journals Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase

2021 ◽  
Vol 9 (4) ◽  
pp. 749
Author(s):  
Gülbahar Abaramak ◽  
Jaime Ricardo Porras-Domínguez ◽  
Henry Christopher Janse van Rensburg ◽  
Eveline Lescrinier ◽  
Ebru Toksoy Öner ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Phylogenetic Analyses ◽  
Halophilic Archaea ◽  
Maximum Activity ◽  
Physiological Relevance ◽  
Health Related ◽  
Specific Alternative ◽  
Binding Groove ◽  
Native Host

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

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