Identification and characterization of the Bacillus thuringiensis phaZ gene, encoding new intracellular poly-3-hydroxybutyrate depolymerase.
A gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome of Bacillus thuringiensis subsp. israelensis ATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designated phaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD of Pseudomonas putida was unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. The B. thuringiensis PhaZ was inactive against p-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-type B. thuringiensis cells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from a phaZ mutant lost this activity. The phaZ mutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S(102)-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate that B. thuringiensis harbors a new type of intracellular PHB depolymerase.
Tseng
Chi-Ling
CL
Institute of Biochemistry and Molecular Biology, School of Life Science, National Yang-Ming University, Taipei 112, Taiwan, Republic of China.
Chen
Hui-Ju
HJ
Shaw
Gwo-Chyuan
GC
D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D045424:Complex Mixtures / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D010168:Palmitates / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D013379:Substrate Specificity; D024363:Transcription Initiation Site; D014280:Triglycerides / Q000378:metabolism; D014304:Triolein / Q000378:metabolism; D000094122:Polyhydroxybutyrates
D002265:Carboxylic Ester Hydrolases; D045424:Complex Mixtures; D006885:Hydroxybutyrates; D010168:Palmitates; D011091:Polyesters; D021382:Protein Sorting Signals; D014280:Triglycerides; D014304:Triolein; C050015:4-nitrophenyl palmitate; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C022417:poly-beta-hydroxybutyrate depolymerase; C005830:tributyrin