Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system.

摘要: Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, PHB A, respectively. Four of phaZ4) have been cloned, one them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Muller, H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA gene (phaZ5) identified by colony hybridization recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. nucleotide sequence 3,704-bp EcoRI fragment determined found contain two large open reading frames (ORFs) coded for polypeptide significant similarities glycerol-3-phosphate dehydrogenases various sources (313 amino acids; M(r), 32,193) precursor A (PhaZ5; 433 44,906). PHV (phaZ4) subcloned, 3,109-bp BamHI determined. Two ORFs (ORF3 ORF4) that represent putative coding regions were identified. deduced acid ORF3 (134 14,686) revealed branched-chain aminotransferase (IlfE) enterobacteria. ORF4 (1,712 bp) as (567 59,947). Analysis primary structures P. Alcaligenes faecalis pickettii homologies 25 83% each other domain structure: at their N termini, they typical signal peptides exoenzymes. adjacent catalytic domains are characterized several conserved acids constitute triads consist consensus serine-dependent hydrolases including pentapeptide G-X-S-X-G, histidine aspartate, region resembling oxyanion hole lipases. C terminal an approximately 40-amino-acid-long threonine-rich (22 27 threonine residues) is present PhaZ1, PhaZ2, PhaZ3, PhaZ5. Instead PhaZ4 A. 90-amino-acid-long fibronectin type III module eucaryotic extracellular matrix proteins. function remains obscure. types C-terminal sequences apparently substrate-binding sites; PhaZ5 PHV-hydrolyzing (PhaZ4 PhaZ1). phaZ1 transferred eutrophus H16 JMP222. All transconjugants both strains able grow carbon source produced translucent halos on PHB-containing solid media. PhaZ4, purified from E. coli; processing sites precursors same lemoignei, similar substrate specificities wild-type hydrolyzed high specific activities. PhaZ1 additionally cleaved PHV, poly(4-hydroxybutyrate). None hydrolyze polyactide or consisting monomers more than atoms. While proteins glycosylated glucose N-acetylglucosamine, none glycosylated. hydrolysis dependent divalent cations such Ca2+ inhibited presence EDTA.