Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene.
An extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Pseudomonas sp. strain A1. The purified enzyme hydrolyzed the D-(-)-3-hydroxybutyrate dimer and trimer at similar rates. The enzyme activity was inhibited by a low concentration of diisopropylfluorophosphate. The molecular weight of the hydrolase was estimated to be about 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 10-kbp DNA fragment of A1 was detected by hybridization with the gene (2 kbp) of an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Subsequent subcloning showed that a SmaI-KpnI fragment (2.8 kbp) was responsible for expression of the hydrolase in Escherichia coli and an in vitro transcription-translation system. The expressed protein detected by immunostaining had the same molecular weight as the purified enzyme from A1. The protein band detected in the in vitro transcription-translation system had a molecular size of 72 kDa. The nucleotide sequence of the SmaI-KpnI fragment was determined, and one open reading frame (2,112 nucleotides) was found. It specifies a protein with a deduced molecular weight of 72,876 (704 amino acids). In this sequence, the consensus sequence of serine-dependent hydrolysis, G-X-S-X-G, did not exist.
Zhang
K
K
Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Japan.
Shiraki
M
M
Saito
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006885:Hydroxybutyrates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011091:Polyesters / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D000094122:Polyhydroxybutyrates
D002265:Carboxylic Ester Hydrolases; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C032152:hydroxybutyrate-dimer hydrolase