|Gene:||phaZ7||Accession Number: G-12287 (MetaCyc)|
Species: Paucimonas lemoignei
The apparent molecular mass of the native enzyme was 36 kDa by SDS-PAGE and 36.199 kDa by MALDI-TOF mass spectrometry [Handrick01].
Polyhydroxyalkanoates (PHAs) are a class of bacterial storage compounds that accumulate inside cells in the form of inclusion bodies (granules) during unbalanced growth conditions. a poly-β-hydroxybutyrate (PHB) is the most abundant storage compound in bacteria (see pathway polyhydroxybutyrate 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 22.214.171.124) or medium-chain-length (EC 126.96.36.199) 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]).
The PhaZ7 extracellular depolymerase from the PHA-degrading bacterium Paucimonas lemoignei (previously known as Pseudomonas lemoignei) hydrolyzes short-chain-length PHAs. The native enzyme was purified from the culture fluid of this organism. It was found to be unusual in its specificity for the native (amorphous) type of PHB substrate which is typical of intracellular depolymerases, and its inactivity toward the denatured type of PHB substrate which is typically the substrate for extracellular PHA depolymerases [Handrick01]. Six other extracellular PhaZ depolymerases have been identified in this organism, all of which are typical in their specificity for the denatured type of PHB substrate (in [Handrick01]).
The crystal structure of this enzyme has been determined at 1.90 Å [Papageorgiou08] and 1.2 Å [Wakadkar10] resolution. The protein was shown to be a monomer, although a dimer was found in the crystal structure which was suggested to be a result of crystal packing [Papageorgiou08].
This enzyme represents the first member of a new subgroup (EC 188.8.131.52) of serine hydrolases which have no significant amino acid sequence similarities with other PHB depolymerases, lipases or hydrolases with the exception of several short regions of sequence. The enzyme was determined to be a serine hydrolase with a Ser-Asp-His catalytic triad and an oxyanion pocket (in [Kapetaniou05]). Site-directed mutagenesis of phaZ7 followed by heterologous expression in, and efficient secretion from, a protease-deficient strain of Bacillus subtilis identified the amino acid residues important to catalysis [Braaz03].
Assays for this and other PHA depolymerase enzymes have been developed based on either neutralization of a 3-hydroxyalkanoate and/or its oligomers produced during hydrolysis, or derivatization of the hydrolysis products and their identification by HPLC (up to an octamer in chain length) [Gebauer06].
Molecular Weight of Polypeptide: 40.02 kD (from nucleotide sequence), 36.0 kD (experimental) [Handrick01 ]
Enzymatic reaction of: poly(3-hydroxybutyrate) depolymerase
Synonyms: poly[(R)-3-hydroxybutyrate] hydrolase
EC Number: 184.108.40.206
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
The reaction is physiologically favored in the direction shown.
This PHB depolymerase was shown to be a serine hydrolase that is thermotolerant and active at alkaline pH. The enzyme used native (amorphous) granules of natural PHB or poly[(R)]-3-hydroxyvalerate as substrate. It did not utilize any denatured, partially crystalline PHA as substrate, or any medium-chain-length PHA such as native poly-3-hydroxyoctanoate granules. It had very low or no activity with a variety of other substrates tested [Handrick01].
T(opt): 65 °C [Handrick01]
pH(opt): 9.5-10 [Handrick01]
Braaz03: Braaz R, Handrick R, Jendrossek D (2003). "Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei." FEMS Microbiol Lett 224(1);107-12. PMID: 12855176
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
Wakadkar10: Wakadkar S, Hermawan S, Jendrossek D, Papageorgiou AC (2010). "The structure of PhaZ7 at atomic (1.2 A) resolution reveals details of the active site and suggests a substrate-binding mode." Acta Crystallogr Sect F Struct Biol Cryst Commun 66(Pt 6);648-54. PMID: 20516591
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