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  • 1
    Online Resource
    Online Resource
    Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases
    American Society for Microbiology ; 2001
    In:  Journal of Bacteriology Vol. 183, No. 17 ( 2001-09), p. 5058-5066
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 183, No. 17 ( 2001-09), p. 5058-5066
    Abstract: Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli , and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k cat and K m values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s −1 and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK a of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.183.17.5058-5066.2001
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2001
    detail.hit.zdb_id: 1481988-0
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  • 2
    Online Resource
    Online Resource
    Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding an α-Amino Acid Ester Hydrolase from Acetobacter turbidans
    American Society for Microbiology ; 2002
    In:  Applied and Environmental Microbiology Vol. 68, No. 1 ( 2002-01), p. 211-218
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 68, No. 1 ( 2002-01), p. 211-218
    Abstract: The α-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing β-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified α-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA , encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli . The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus , but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the α-amino acid ester hydrolase is a β-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The α-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of β-lactam antibiotic acylases.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.68.1.211-218.2002
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2002
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    Enterococcus faecalis strains show culture heterogeneity in cell surface charge
    Microbiology Society ; 2006
    In:  Microbiology Vol. 152, No. 3 ( 2006-03-01), p. 807-814
    Details
    In: Microbiology, Microbiology Society, Vol. 152, No. 3 ( 2006-03-01), p. 807-814
    Abstract: Adhesion of micro-organisms to biotic and abiotic surfaces is an important virulence factor and involves different types of interactions. Enterococcus faecalis , a human commensal and an important opportunistic pathogen, has the ability to adhere to surfaces. Biliary stents frequently become clogged with bacterial biofilms, with E. faecalis as one of the predominant species. Six E. faecalis strains isolated from clogged biliary stents were investigated for the presence of specific biochemical factors involved in their adhesion: aggregation substances (Aggs) and the enterococcal surface protein (encoded by the esp gene). In addition, physico-chemical factors involved in adhesion (zeta potential and cell surface hydrophobicity) were determined, as well as the influence of ox bile on these properties. Two-thirds of the biliary stent isolates displayed culture heterogeneity in the pH dependence of their zeta potentials. Moreover, 24 out of 46 clinical isolates of E. faecalis , including 11 laboratory strains, also displayed such heterogeneity. The culture heterogeneity was demonstrated to be a stable trait, not caused by quorum sensing, not plasmid mediated, and independent of the presence of esp and Agg. Data presented show that culture heterogeneity in zeta potential enhances adhesion to an abiotic surface. A higher prevalence of culture heterogeneity in zeta potential in pathogenic as compared to non-pathogenic isolates could indicate that this phenomenon might play a role in virulence and putatively in pathogenesis.
    Type of Medium: Online Resource
    ISSN: 1350-0872 , 1465-2080
    URL: Article
    DOI: 10.1099/mic.0.28460-0
    Language: English
    Publisher: Microbiology Society
    Publication Date: 2006
    detail.hit.zdb_id: 2008736-6
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    Influence of Culture Heterogeneity in Cell Surface Charge on Adhesion and Biofilm Formation by Enterococcus faecalis
    American Society for Microbiology ; 2006
    In:  Journal of Bacteriology Vol. 188, No. 7 ( 2006-04), p. 2421-2426
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 188, No. 7 ( 2006-04), p. 2421-2426
    Abstract: Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms. Enterococcus faecalis strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of E. faecalis strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein ( esp ) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.188.7.2421-2426.2006
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Steady-State Kinetics and Tryptophan Fluorescence Properties of Halohydrin Dehalogenase from Agrobacterium radiobacter . Roles of W139 and W249 in the Active Site and Halide-Induced Conformational Change
    American Chemical Society (ACS) ; 2003
    In:  Biochemistry Vol. 42, No. 47 ( 2003-12-01), p. 14057-14065
    Details
    In: Biochemistry, American Chemical Society (ACS), Vol. 42, No. 47 ( 2003-12-01), p. 14057-14065
    Type of Medium: Online Resource
    ISSN: 0006-2960 , 1520-4995
    URL: Article
    DOI: 10.1021/bi034941a
    RVK:
    WA 15000
    Language: English
    Publisher: American Chemical Society (ACS)
    Publication Date: 2003
    detail.hit.zdb_id: 1472258-6
    SSG: 12
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