Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene.
The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53,095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.
Jendrossek
D
D
Institut für Mikrobiologie der Georg-August-Universität Göttingen, Germany.
Backhaus
M
M
Andermann
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase