HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 7, 4951-4962, February 16, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/7/4951    most recent M608119200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Eugenio, L. I. Articles by Prieto, M. A. Search for Related Content PubMed PubMed Citation Articles by de Eugenio, L. I. Articles by Prieto, M. A. Social Bookmarking                      What's this?

Biochemical Evidence That phaZ Gene Encodes a Specific Intracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase in Pseudomonas putida KT2442

CHARACTERIZATION OF A PARADIGMATIC ENZYME^*

Laura I. de Eugenio, Pedro García, José M. Luengo, Jesús M. Sanz^¶, Julio San Román^||, José Luis García, and María A. Prieto¹

From the Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), C. Ramiro de Maeztu, 9, 28040 Madrid, the Departamento de Bioquímica y Biología Molecular, Universidad de León, 24007 León, the ^¶Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Av. Universidad, s/n. 03202 Elche (Alicante), and the ^||Instituto de Ciencia y Tecnología de Polímeros, CSIC, C. Juan de la Cierva 3, 28006 Madrid, Spain

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra- or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a core/ hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/exo-mcl-PHA depolymerases.

Received for publication, August 23, 2006 , and in revised form, December 13, 2006.

^* This work was supported by the Comisión Interministerial de Ciencia y Tecnología (Grants GEN2001-4698-C05-02, BIO2003-05309-C04-01/02 and CTM2006-04007) and by the European Union (Grant 6FP-026515-2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Tel.: 34-91-8373112 (ext. 4228); Fax: 34-91-5360432; E-mail: auxi{at}cib.csic.es.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				D. Jendrossek Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes) J. Bacteriol., May 15, 2009; 191(10): 3195 - 3202. [Full Text] [PDF]

				K. Uchino, T. Saito, and D. Jendrossek Poly(3-Hydroxybutyrate) (PHB) Depolymerase PhaZa1 Is Involved in Mobilization of Accumulated PHB in Ralstonia eutropha H16 Appl. Envir. Microbiol., February 15, 2008; 74(4): 1058 - 1063. [Abstract] [Full Text] [PDF]

				K. Uchino, T. Saito, B. Gebauer, and D. Jendrossek Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA J. Bacteriol., November 15, 2007; 189(22): 8250 - 8256. [Abstract] [Full Text] [PDF]