Molecular characterization of the extracellular poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product.
phaZPfi, the gene encoding the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, was cloned, sequenced, and characterized. It comprises 837 bp and is transcribed as a monocistronic message of about 950 bp from a putative sigma 70-like promoter 32 bp upstream of the ATG start codon. The deduced protein of 278 amino acids reveals a typical leader peptide at its N terminus. When expressed in Escherichia coli, the mature depolymerase started with Ala-23, whereas the mature enzyme purified from P. fluorescens GK13 started with both Leu-34 and Arg-35 determining proteins of 26,687 and 26,573 Da, respectively. The depolymerase is a strongly hydrophobic protein and includes the lipase consensus sequence Gly-X-Ser-X-Gly, which is known for serine hydrolases. Replacement of the central residue, Ser-172, in the corresponding sequence (Gly-Ile-Ser-Ser-Gly) of PhaZPfl with alanine resulted in complete loss of enzyme activity, indicating that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.
Schirmer
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Institut für Mikrobiologie der Georg-August-Universität Göttingen, Germany.
Jendrossek
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D005815:Genetic Code; D015698:Genomic Library; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011091:Polyesters / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D011091:Polyesters; D012333:RNA, Messenger; D011994:Recombinant Proteins; C088151:poly(3-hydroxyoctanoic acid); D002265:Carboxylic Ester Hydrolases; C080782:poly(3-hydroxyoctanoic acid) depolymerase