Biochemical evidence that phaZ gene encodes a specific intracellular medium chain length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442: characterization of a paradigmatic enzyme.
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 corealpha/beta 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.
de Eugenio
Laura I
LI
Departamento de Microbiologi´a Molecular, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cienti´ficas (CSIC), C. Ramiro de Maeztu, 9, 28040 Madrid.
Garci A
Pedro
P
Departamento de Microbiologi´a Molecular, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cienti´ficas (CSIC), C. Ramiro de Maeztu, 9, 28040 Madrid.
Luengo
José M
JM
Departamento de Bioqui´mica y Biologi´a Molecular, Universidad de Leo´n, 24007 Leo´n.
Sanz
Jesu S M
JSM
Instituto de Biologi´a Molecular y Celular, Universidad Miguel Herna´ndez, Av. Universidad, s/n. 03202 Elche (Alicante), and the.
Roma N
Julio San
JS
Instituto de Ciencia y Tecnologi´a de Poli´meros, CSIC, C. Juan de la Cierva 3, 28006 Madrid, Spain.
Garci A
José Luis
JL
Departamento de Microbiologi´a Molecular, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cienti´ficas (CSIC), C. Ramiro de Maeztu, 9, 28040 Madrid.
Prieto
Mari A A
MAA
Departamento de Microbiologi´a Molecular, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cienti´ficas (CSIC), C. Ramiro de Maeztu, 9, 28040 Madrid. Electronic address: auxi@cib.csic.es.
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase