Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1

Protein Science : a Publication of the Protein Society
Roberta ScognamiglioAlberto Di Donato

Abstract

A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our resul...Continue Reading

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Citations

Sep 19, 2002·Protein Science : a Publication of the Protein Society·Maria MontiPiero Pucci
Apr 7, 2004·Applied and Environmental Microbiology·Valeria CafaroAlberto Di Donato
Jun 9, 2006·Microbiology and Molecular Biology Reviews : MMBR·Jorge LalucatNorberto J Palleroni
Jul 12, 2005·Proceedings of the National Academy of Sciences of the United States of America·Nicolae DameanGeorge M Whitesides
Sep 29, 2004·Biochemical and Biophysical Research Communications·Jirí SantrůcekMilan Kodícek
Oct 11, 2003·FEMS Microbiology Reviews·Joseph G LeahySuzanne M Morcomb
May 20, 2008·Advances in Applied Microbiology·R E ParalesJ L Ditty

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