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

¹ Dipartimento di Chimica Organica e Biologica, Universitá di Napoli Federico II, 80134 Napoli, Italy
² Dipartimento di Genetica e di Biologia dei Microrganismi, Universitá di Milano, 20133 Milano, Italy
³ Centro Internazionale di Servizi di Spettrometria di Massa, 80131 Napoli, Italy
⁴ IRBM P. Angeletti, Via Pontina Km 30.600—00040 Pomezia (RM), Italy

Reprint requests to: Alberto Di Donato, Dipartimento di Chimica Organica e Biologica, Universitá di Napoli Federico II, Via Mezzocannone, 16. 80134 Napoli, Italy; e-mail: didonato{at}unina.it; fax (39) (081) 552-1217.

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 results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building.

Keywords: Monooxygenase; expression; Pseudomonas; recombinant; limited proteolysis; mass spectrometry

Abbreviations: Tomo, toluene/o-xylene-monooxygenase • Tomo D, subunit D of the complex Tomo • DmpM (protein P2), regulatory component of phenol hydroxylase • ORF, open reading frame • ES/MS, electrospray mass spectrometry • MALDI, matrix assisted laser desorption ionization • LB, Luria-Bertani • EDTA, ethylenediaminetetraacetic acid • PAGE, polyacrylamide gel electrophoresis • SDS, sodium dodecylsulfate • Tris, tris(hydroxymethyl)aminomethane • DTT, dithiothreitol • HPLC, high pressure liquid chromatography • IPTG, isopropyl-ß-D-thiogalacto-pyranoside • PVDF, polyvinyl difluoro • PDB, Protein Data Bank • V-8, V-8 endoproteinase • Asp-N, endoproteinase Asp-N

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