|Gene:||phaZ||Accession Number: G-12288 (MetaCyc)|
Species: Pseudomonas putida KT2442
The subunit structure of this enzyme has not been reported. The apparent molecular mass of the polypeptide was determined by SDS-PAGE [deEugenio07].
Polyhydroxyalkanoates (PHAs) are a class of bacterial storage compounds that accumulate inside cells in the form of inclusion bodies (granules) during unbalanced growth conditions. poly-3-hydroxybutanoate (PHB) is the most abundant storage compound in bacteria (see pathway polyhydroxybutanoate biosynthesis). PHAs may accumulate at levels of up to 90% of cellular dry weight and can provide the bacterium with a source of carbon and energy. These polyesters are of industrial interest due to their biodegradability to water-soluble products, and their ability to be synthesized from renewable resources (in [Papageorgiou08] and in [Handrick01] and reviewed in [Jendrossek09]).
Many 3-hydroxyacids have been identified as constituents of PHA polymers. PHB and its copolymer with 3-hydroxyvalerate have been commercialized. PHAs are generally classified as short-chain-length (monomers contain 3-5 carbon atoms) or medium-chain-length (monomers contain six or more carbon atoms) (in [Kapetaniou05]).
Enzymes that degrade PHAs can be of two types, those that are secreted and act extracellularly, or those that act intracellularly. They may be specific for either short-chain-length (EC 18.104.22.168) or medium-chain-length (EC 22.214.171.124) PHAs and catalyze their hydrolysis to monomeric, or oligomeric hydroxyalkanoates. Intracellular enzymes degrade PHAs for direct utilization, while extracellularly secreted enzymes may act on PHAs that are released from dead bacterial cells (in [Kapetaniou05] and in [Papageorgiou08]).
Intracellular medium-chain-length polyhydroxyalkanoate depolymerases have been identified in members of the genus Pseudomonas. These phaZ genes are encoded within the pha gene cluster in these organisms. The PhaZ enzyme from Pseudomonas putida KT2442 is the first biochemically characterized intracellular medium-chain-length polyhydroxyalkanoate depolymerase. Mutagenesis and complementation evidence for phaZ gene function has been presented for Pseudomonas putida GPo1 (formerly Pseudomonas oleovorans GPo1), Pseudomonas putida U, and Pseudomonas putida KT2442 (a derivative of the parental strain Pseudomonas putida KT2440) [deEugenio07]. This enzyme has been shown to be involved in the PHA turnover cycle in this organism [deEugenio10].
Recombinant His-tagged enzyme was overexpressed in Escherichia coli, purified, and characterized. It was shown to be a serine hydrolase. The enzyme was also shown to be a PHA granule-associated protein [deEugenio07].
Molecular Weight of Polypeptide: 31.74 kD (from nucleotide sequence), 33.0 kD (experimental) [deEugenio07 ]
Unification Links: Protein Model Portal:Q8KQ22
Enzymatic reaction of: poly(3-hydroxyoctanoate) depolymerase
Synonyms: intracellular medium chain length polyhydroxyalkanoate depolymerase
EC Number: 126.96.36.199
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.
The reaction is physiologically favored in the direction shown.
The enzyme required a high ionic strength (300 mM sodium chloride or potassium chloride) for optimal activity. It was inhibited by several ionic and nonionic detergents, although cholate did not inhibit [deEugenio07].
A sensitive radioactive assay was developed for detection of PHA hydrolysis in vitro in crude extracts using a [14C] labeled copolymer of hydroxyoctanoate and hydroxyhexanoate in a polymer/water emulsion. These native polymer granules were produced in vivo in a different strain of P. putida. A less sensitive, non-radioactive, turbidometric assay could be used for the purified enzyme [deEugenio07].
The purified enzyme was similarly active on both native and artificially produced granules and was specific for hydrolysis of medium-chain-length PHAs containing aliphatic and aromatic monomers. It showed no activity with poly-3-hydroxybutanoate as substrate and did not hydrolyze various triacylglycerols or p-nitrophenylalkanoates, which are lipase or esterase substrates. Monomeric and dimeric products of PHA hydrolysis were identified by LC-MS. The enzyme appeared to function as both an endo- and exo-hydrolase [deEugenio07].
T(opt): 43 °C [deEugenio07]
pH(opt): 8.8 [deEugenio07]
deEugenio07: de Eugenio LI, Garcia P, Luengo JM, Sanz JM, Roman JS, Garcia JL, Prieto MA (2007). "Biochemical evidence that phaZ gene encodes a specific intracellular medium chain length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442: characterization of a paradigmatic enzyme." J Biol Chem 282(7);4951-62. PMID: 17170116
deEugenio10: de Eugenio LI, Escapa IF, Morales V, Dinjaski N, Galan B, Garcia JL, Prieto MA (2010). "The turnover of medium-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance." Environ Microbiol 12(1);207-21. PMID: 19788655
Handrick01: Handrick R, Reinhardt S, Focarete ML, Scandola M, Adamus G, Kowalczuk M, Jendrossek D (2001). "A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids." J Biol Chem 276(39);36215-24. PMID: 11457823
Jia01: Jia Y, Yuan W, Wodzinska J, Park C, Sinskey AJ, Stubbe J (2001). "Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism." Biochemistry 40(4);1011-9. PMID: 11170423
Kapetaniou05: Kapetaniou EG, Braaz R, Jendrossek D, Papageorgiou AC (2005). "Crystallization and preliminary X-ray analysis of a novel thermoalkalophilic poly(3-hydroxybutyrate) depolymerase (PhaZ7) from Paucimonas lemoignei." Acta Crystallogr Sect F Struct Biol Cryst Commun 61(Pt 5);479-81. PMID: 16511073
Papageorgiou08: Papageorgiou AC, Hermawan S, Singh CB, Jendrossek D (2008). "Structural basis of poly(3-hydroxybutyrate) hydrolysis by PhaZ7 depolymerase from Paucimonas lemoignei." J Mol Biol 382(5);1184-94. PMID: 18706425
©2014 SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493