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Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering. Hikaru Suenaga, 2002.Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation step during the metabolism of biphenyl . The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds, including polychlorinated biphenyls (PCBs) . Based on crystallographic analyses of naphthalene dioxygenase (B . Kauppi, K . Lee, E . Carredano, R . E . Parales, D . T . Gibson, H . Eklund, and S . Ramaswamy, Structure 6:571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of Pseudomonas pseudoalcaligenes KF707 . Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in Escherichia coli . The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox . In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2',5'-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2'-trichlorobiphenyl and 2,5,4'-trichlorobiphenyl . Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2',3'-dihydroxy biphenyl with concomitant dechlorination . In Situ Accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains. João Inácio, 2003.Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms . However, species-specific oligonucleotide probes often fail to give satisfactory results . Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons . In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455T . The brightest signal was obtained with a probe complementary to positions 223 to 240 . Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells . The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes) . This work contributes to a more rational design of species-specific probes for yeast identification and monitoring .
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