Similar enzymes, different structures: Phthalate dioxygenase is an α3α3 stacked hexamer, not an α3β3 trimer like “normal” Rieske oxygenases

Abstract

Phthalate dioxygenase is an α 3 α 3 stacked hexamer, not an α 3 β 3 trimer like "normal" Rieske oxygenases.Michael Tarasev AbstractPhthalate dioxygenase (PDO) is a member of a class of bacterial oxygenases that contain both Rieske [2Fe-2S] and Fe(II) mononuclear centers. Recent crystal structures of several Rieske dioxygenases showed that they exist as α 3 β 3 multimers with subunits arranged head-to-tail in α and β stacked planar consists of only α-subunits, remains to be solved. Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ in the mechanism of catalysis. Gel filtration and analytical centrifugation experiments, supplemented with mass spectrometric analysis (both ESI-MS and ESI-GEMMA), in this work showed a hexameric arrangement of subunits in the PDO multimer. Our proposed model for the subunit arrangement in PDO postulates two α 3 planar rings one on top the other, similar to the α 3 β 3 arrangement in other Rieske dioxygenases. Unlike other Rieske dioxygenases, this arrangement brings two Rieske and two mononuclear centers, all on separate subunits, into proximity, allowing their cooperation for catalysis. Potential reasons necessitating this unusual structural arrangement are discussed. KeywordsPhthalate Dioxygenase; Rieske center; Rieske dioxygenase; structure; subunit arrangement Rieske aromatic ring-hydroxylating dioxygenase systems facilitate the degradation of various aromatic hydrocarbons. These enzymes contain Rieske-type redox-active iron-sulfur clusters ) and mononuclear ferrous centers, and catalyze the formation of cis-dihydrodiols by enantiospecific addition of two hydroxyl groups onto aromatic substrates. This is a first step in many aerobic biodegradation pathways that typically lead to breaching of aromatic rings of otherwise recalcitrant substrates.Phthalate dioxygenase (PDO), an oxygenase from Burkholderia cepacia (DB01), catalyzes the first step in the breakdown of the aromatic compound phthalate to form the dihydrodiol of * To whom correspondence should be addressed. Email: dballou@umich.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. . It is a member of the Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ somewhat in its mechanism of catalysis. For example, after reduced NDO is exposed to oxygen in the presence of the substrate, the mononuclear iron was found to be in the ferric state [6]. One cis-dihydrodiol product was found per Rieske and mononuclear center that was oxidized. NIH Public AccessIn contrast, upon completion of the reaction in PDO, phthe mononucle...

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“…The inset figures show the zero charge deconvolution mass spectrum. (Figure adapted from Reference with permission from the publisher. )…”

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